CARIBBEAN REGIONAL STANDARD OPERATING PROCEDURES

Media Preparation and Quality Control – SOP No: CRM-SOP 18

CAREC PAHO WHO EUROPEAN UNION

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Media Preparation and Quality Control

Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 1 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

TABLE OF CONTENTS

Amendment procedure 4 Introduction 5 Media production process flow chart 5 1. Media Preparation and Quality Control 6 1.1 Introduction 6 1.2 Types of Media 6 1.3 Media Production 10 2.0 Media Quality Control Methods 11 2.1 Introduction 11 2.2 Principles of QC of Media 11 2.3 Shelf-life 11 2.4 Standardization of Laboratory Media 12 2.5 Control Medium 12 2.6 Test Programme 12 2.7 Physical Characteristics 12 2.8 Quality documentation 12 2.9 Methods 12 2.10 Examination of Physical Characteristics 13 2.11 Test for Microbiological Performance 14 2.12 QC Tests for Solid Media 14 2.13 QC Tests for Liquid Media 15 2.14 Interpretation of Microbiological Tests on Liquid Media 15 2.15 Sterility Tests 15 2.16 Quality Control Limits for Haemophilus Test Medium Agar 15 2.17 Storage of Culture Media 16 3.0 Quality Control of Culture Media Prepared In-house 16 3.1 Procedure 16 3.2 Quality Control Protocols 16 4.0 Quality Control of Commercially Prepared Media 18 4.1 Exempt Media Category 18 4.2 Nonexempt Media Category. 18 4.3 General QC Requirements for both Exempt and Nonexempt Media 19 4.4 Q.C. Requirements for Nonexempt Media Only 19 4.5 Reporting Deficiencies Identified by the User 19 5.0 Quality Control of Media not included in Section 6 20 6.0 Methods 24 6.1 Preparation of Alkaline Peptone Water 24 6.2 Preparation of Blood Agar 26 6.3 Preparation of 28 6.4 Preparation of Baird Parker Agar 30 6.5 Preparation of Bile Esculin Azide Agar 31 6.6 Preparation of 34 6.7 Preparation of CLED Agar 36

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Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 2 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

6.8 Preparation of Cooked Meat Medium 38 6.9 Preparation of Brain Heart Infusion Broth 40 6.10 Preparation of Brain Heart Infusion Agar 42 6.11 Preparation of GN Broth 44 6.12 Preparation of Haemophilus Test Medium (HTM) Agar 46 6.13 Preparation of MacConkey Agar 48 6.14 Preparation of MacConkey Agar with Sorbitol 50 6.15 Preparation of Mueller Hinton (MH) Agar 52 6.16 Preparation of 54 6.17 Preparation of New York City (NYC) Medium 56 6.18 Preparation of Peptone Water 58 6.19 Preparation of Phenylethyl Alcohol (PEA) Agar 60 6.20 Preparation of Phenylalanine Agar 62 6.21 Preparation of (MSA) 64 6.22 Preparation of Sabouraud Dextrose Agar 66 6.23 Preparation of Thayer Martin (TM) Agar 68 6.24 Preparation of Xylose Lysine Deoxycholate (XLD) Agar 70 6.25 Preparation of Thiosulphate Citrate Bile Salt Sucrose (TCBS) Agar 72 6.26 Preparation of Trichosel Broth 74 6.27 Preparation of Tetrathionate Broth 76 6.28 Preparation of Candida Bromcresol Green (BCG) Agar 78 6.29 Preparation of CHROMagar 80 6.30 Preparation of Lim Broth 82 6.31 Preparation of Kligler Iron Agar (KIA) 84 6.32 Preparation of Urea 86 6.33 Preparation of Simmons Citrate 88 6.34 Preparation of MILS Medium 90 6.35 Preparation of Triple Sugar Iron (TSI) Agar 93 7.0 References 96 Appendix 1: Preparation of Media (Flowchart) 97 Appendix 2: Preparation of Suspensions for Quality Control of Nonexempt Media 98 Appendix 3: Media Quality Control Record Sheet 99 Appendix 4: Media Quality Control Sheet 100 Appendix 5: Exempt and Non-exempt Categories for Media 102

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN 2 A CARIFORUM Project Funded by the European Union and Implemented by CAREC Media Preparation and Quality Control

Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 3 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

Acknowledgements The ‘Strengthening of Medical Laboratory Services in the Caribbean’ (SMLS) Project, would like to thank the following people for their participation in researching and writing the microbiology standard methods, and also the individual countries for supporting this initiative.

Many thanks also for the facilitators of the standard methods meetings, the method editors and administrative support.

Participants

Country Name Organisation Aruba Ms. Astrid Dirksz Landslaboratorium Anguilla Ms. Everette Duncan Princess Alexandra Hospital, Stoney Ground Bahamas Mr. Allison Scavella Princess Margaret Hospital Barbados Ms. Gail Trotman Public Health Laboratory Ms. Juliana Applewaite Queen Elizabeth Hospital Mr. Edmund Blades Public Health Laboratory Belize Ms. Solitaire Parra Maza Central Medical Laboratory British Virgin Islands Ms. Allene Brewley Peebles Hospital Ms. June Greene Medicure Ltd. Ms. Wayveney Armstrong MEDICAL Diagnostic Laboratory Cayman Islands Mr. Dale Andrew Chin Caymans Islands Health Services Authority Curacao Mr. Osric Wanga Analytical Diagnostic Centre Ms. Helga Leito Analytical Diagnostic Centre Grenada Ms. Sonia Ann Edwards St Georges Hospital Guyana Ms. Alexis Wilson Pearson Georgetown Public Hospital Ms. Ede Tyrell Langevine University of Guyana Jamaica Ms. Rayaad Khan Central Medical Laboratories Ltd Ms. Valerie Levy UWI Microbiology Department Ms. Heather Wint Caledonia Medical Laboratory (Biomedical) Ms. Adriene Kellier Cornwall Regional Hospital Mr. Norman S. Burke National Public Health Laboratory St Lucia Mr. Martin S. Mc Kenzie Ezra Long Laboratory St Vincent and Mr. Elliot Samuel Milton Cato Memorial Hospital the Grenadines Caribbean Epidemiology Ms. Radha Gosein CAREC Centre (CAREC) Ms. Michele Nurse-Lucas CAREC Ms. Denise Clarke CAREC Dr. Ashok Rattan CAREC Trinidad Ms. Zobida Khan Mohammed Trinidad Public Health Laboratory (TPHL) Mr. Anthony Bayley Trinidad Public Health Laboratory (TPHL) Mr. Jawaheerlal Mewahlal College Of Science, Technology And Applied Arts of Trinidad and Tobago (COSTAATT) Ms. Monica Pollard Port of Spain General Hospital Dr. William Swanston Eric Williams Sciences Medical Complex Turks and Caicos Islands Ms. Lessonjule Lyons Doney Grand Turk General Hospital Laboratory Ms. Peggy Hermitt Myrtle Rigby Hospital

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Facilitators/Editors

Country Name Organisation Canada Dr Harold Richardson Quality Management Program – Laboratory Section Trinidad Ms. Julie Sims Strengthening of Medical Laboratory Services in the Caribbean (SMLS) United Kingdom Ms. Jacki Watts Bristol Health Protection Agency United Kingdom Ms. Valerie Bevan Health Protection Agency (HPA UK)*

Administrative Support

Function Name Organisation Administrative Support Ms. Stacey-Mae Stephens SMLS Project Administrative Support Ms. Reesa Moonsie SMLS Project Rapporteur Ms. Margaret Hunte Independent

Graphic Design and Support

Function Name Country Graphic Design Ms. Cathleen Jones Trinidad & Tobago Graphic Support Mr. Adrian Nicholls United Kingdom Graphic Support Ms. Karen Lara-Augustine Trinidad & Tobago

*The Health Protection Agency, England, is acknowledged for the knowledge and expertise of their staff and working groups in the UK who develop National Standard Methods, on which the Caribbean Regional Microbiology Standard Operating Procedures are based.

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Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 5 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

INTRODUCTION

The Caribbean Regional Standard Methods include a selection will be based on a review of EQA results, most variety of standard, validated methods, produced as common and/or critical tests. Part of the method a single standard operating procedure (SOP) for use in a standardization process will be an ongoing review and variety of levels of microbiology laboratory service. amendment procedure. The CSMDG consists of It is intended that these methods provide detailed microbiology laboratory representatives from most of instructions for microbiology services for microbiological the CARIFORUM countries, all of whom were nominated investigations, in order to provide accurate, reliable and to the task by the CRMC. reproducible results which will have clinical utility. These methods may be adopted by laboratories within the This initiative should enable the region to implement a region, or adapted, provided that such adaptations use standardized and constructive method for ensuring that an evidence-based validation process. validated methods are available for the region, and that they are updated as required. These methods have been developed by the Caribbean Regional Microbiology Standard Methods Drafting Advantages of using regionally validated methods are Group (CSMDG) in response to a request by the to improve quality, make better use of resources, Caribbean Regional Microbiology Council (CRMC), reduce costs, enable central procurement & media which was set up by the CARIFORUM Project entitled preparation, facilitate staff training and transfers due to ‘Strengthening of Medical Laboratories in the horizontal integration, a reduction in variability of service Caribbean’ to strengthen specifically the microbiology provision, an improved quality of surveillance data, services in the Caribbean Region. The Project was and the purchase of appropriate equipment. A major initiated in response to findings which indicated that advantage is that the availability of regional standard there was an unacceptable level of error in laboratories methods would assist microbiology laboratories with within the region. External quality assessment results documentation for accreditation revealed that microbiology laboratories were not performing well and feedback from the region via Although the CSMDG has taken every care with the laboratory staff, lab managers and directors was that preparation and issue of these standard procedures, they felt that guidance in microbiology requirements was and they have been validated regionally, nationally and required. internationally, the CSMDG, or any other organization, cannot be responsible for the accuracy of any statement The background for this initiative is a worldwide move to or representation made or the consequences arising implement standards in all areas, which has now from the use of or alteration to any information extended to include medical laboratories. As tourism is contained in them. These procedures are intended so vital to the region’s economy, the need for accurate solely as a resource for practicing microbiology diagnosis and treatment is paramount. It was accepted professionals in the field, operating in the Caribbean that there is a requirement for validated methods for region, and specialist advice should be obtained where accreditation purposes and providing validated standard necessary. If changes are made to the original methods will assist in the move towards accreditation. publication, it must be made clear where changes have been made to the original document. When referring to The methods will be chosen for standardization by the these SOPs in successive documentation, the CSMDG Caribbean Regional Microbiology Council, and this should be acknowledged.

STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN 5 A CARIFORUM Project Funded by the European Union and Implemented by CAREC Media Preparation and Quality Control

Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 6 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

Amendment procedure

Controlled Document Reference CRM-SOP 18

Controlled Document Title Standard Operating Procedure for Media Preparation and Quality Control

Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting Group. Any amendments should be validated and authorized by an agreed process, and referenced.

Each Regional Standard Method has an individual record of amendments. The current amendments are listed on this page.

On issue of revised or new pages, each controlled document should be updated by the copyholder in the laboratory.

Amendment Issue Number Insert Issue Page Section(s) Amendment Number/Date Discarded Number involved

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Media production process flow chart

Prepare media according to manufacture specification or using another standardised method

Dispense

Sterility check quality control

Pass fail

Media stored and Media discarded and issued for use problem investigated

Process recorded

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Title: Media Preparation and Quality Control SOP Number: CRM-SOP: 18 Version: 1 Page Number: 8 of 102 Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008

1. Media Preparation and Quality Control 1.1 Introduction Culture media is vital to microbiology for the production of reliable laboratory results. Many components optimize the growth of microorganisms on media. The basic requirements for a medium include a nutrient source, a solidifying agent (for solid media), a specific pH and any number of specific additives. The nutritional requirements of most microorganisms are complex. Most utilize an array of nutrient sources including nitrogen, carbon, inorganic salts, minerals, and other diverse substances. While some organisms can utilize a very simple medium such as nitrate or ammonia, most require protein hydrolysates or peptone. Other selective agents such as antibiotics, dyes, and other nutrient sources, can be incorporated into media for the of a particular organism.

Other considerations that allow optimal microorganism growth include incubation temperature and gas in the growth environment. Most clinically significant organisms are mesophiles, and will optimally grow at temperatures of between 25º and 40ºC. Although most species grow optimally in ambient air; some require CO2 or the total removal of O2.

Liquid media contain all the ingredients described above but contain less of the solidifying agent.

The preparation of culture media from dehydrated media requires accuracy and attention to preparation. For successful and reproducible preparation of culture media, points to be considered include the dehydrated media and ingredients, glassware/plastic ware, equipment, water, dissolving the medium, sterilization, adding enrichments and supplements, pH, dispensing the media, storage and quality control. QC media must be sterile and must support the growth of the microorganism it is intended to isolate.

Commercially produced culture media will have passed extensive process and end point control tests before release. In the laboratory all media undergo reconstitution, heat-processing and, perhaps, supplementation with additives, so it is essential to have quality control to check the effects of these processes.

Sterilization is any process or procedure designed to entirely eliminate viable microorganisms from a material or medium. Sterilization can be accomplished by the use of heat, chemicals, radiation or filtration. A temperature range of 121–124ºC for 15 minutes is an accepted standard condition for sterilizing up to one litre of culture medium. Dry heat is employed for materials such as metal instruments that could be corroded by moist heat. Chemical sterilization is not employed in the preparation of culture media.

Radiation sterilization is an optimal treatment for heat sensitive materials. This includes ultraviolet light and ionizing radiation. Ultraviolet light is chemically active and causes excitation of atoms within the microbial cells, particularly the nucleic acids, producing lethal mutation, and stops the organism from reproducing.

Filtration is a useful method for sterilizing liquids and gases, as it excludes microorganisms rather than destroying them. Two major types of filters may be used, depth filters which entrap particles and membrane filters which screens out particles.

1.2 Types of Media The following are examples of the different types of media that are used for isolation of microorganisms. 1. Transport Media and Preservatives 2. General Purpose, Enriched, Selective, Differential 3. Susceptibility Media 4. Anaerobic Media

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In order to be successful in the isolation of microorganisms sought, the culture media must provide all essential nutrients, ions and moisture, maintained at the correct pH and osmotic pressure, and neutralize any toxic material produced.

1.2.1 Transport Media and Preservatives Transport media are used in the collection and transport of specimens and were initially devised because fastidious organisms would not survive transport from the bedside to inoculation in the laboratory.

Generally, transport media provide a non-nutrient source that sustains the viability of both aerobic and anaerobic organisms without allowing significant growth. Most transport media have specific ingredients that accomplish these goals. These include a small amount of agar to allow a solid base to which the organisms can attach and to reduce desiccation, an indicator oxidation-reduction agent, which shows when oxidation has occurred, and reagents that maintain the pH. Other additives allow the survival of specific organisms e.g. sodium thioglycollate for anaerobes or charcoal which reduces the effect of toxic metabolic products and thus enhances the growth of the pathogen. Other ingredients are added for specific purposes.

Transport media generally allow stability of specimens for 6–12 hours at ambient temperatures and should not be refrigerated since some organisms do not survive at colder temperatures.

Parasitology transport media are actually preservatives meant to maintain the integrity of the parasite and not to maintain viability.

1.2.2 General Purpose General-purpose media are those media capable of detecting most aerobic and facultative anaerobic organisms. Example: sheep’s blood agar, which is used for the general isolation of organisms directly from primary specimens inoculated onto the agar.

1.2.3 Enriched Enriched media are media that allow fastidious organisms to grow because of the presence of specific nutrient additives such as haemin. Fastidious organisms may not grow well on general media. Example: chocolate agar.

1.2.4 Selective Selective media contain additives that enhance the presence of the desired organism by inhibiting other organisms. Most commonly, selection is attained with a dye or with the addition of an antibiotic. An example is crystal violet containing MacConkey agar which inhibits most gram-positive organisms. The effectiveness of selectivity varies and is not always complete, thus partial breakthrough growth or smaller colonies of the inhibited organisms will grow. The ingredients that make the medium highly selective may actually inhibit the desired pathogen for example a medium that is selective for Neisseria gonorrhoeae (GC) and that contains vancomycin may inhibit some strains of vancomycin sensitive GC.

1.2.5 Differential Differential media aid in the presumptive identification of organisms based on the organism’s appearance on the medium. Examples are MacConkey, Hektoen enteric, and xylose-lactose-desoxycholate (XLD) agars used in the isolation of enteric pathogens. On MacConkey agar a bright pink magenta colour colony indicates that is utilizing a lactose fermenter.

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1.2.6 Susceptibility Media Most media use hydrolysate of casein and beef extract since these components are low in thymidine and thyamine contents. Excess amount of thymidine and thymine can make organisms appear to be more susceptible to sulfonamides and trimethoprim. Calcium and magnesium ion concentrations are adjusted to allow correct interpretations of Pseudomonas spp susceptibility results with the aminoglycosides, colistin and tetracycline.

1.2.7 Anaerobic Media All general-purpose nonselective anaerobic blood agar media have similar formulations and include peptones, yeast extract, vitamin K (which is required for some Porphyromonas spp), hemin (enhances the growth of some Bacteroides spp), 5% sheep blood agar (allows for the detection of haemolysis), and reducing agents. All allow the isolation and cultivation of both strictly anaerobic and fastidiously anaerobic organsisms. The difference in each of the media is the small variation in the peptones used and the inclusion of dextrose in some media as an energy source. Additives used with some of these media allow the media to have both selective and differential properties.

1.3 Media Production 1.3.1 Measurement of pH The exact method used to measure pH largely depends on the type of equipment available in the laboratory. 1The pH of the agar should be taken at room temperature after gelling and can be checked by one of the following means: • Macerate a sufficient amount of agar to submerge the tip of a pH electrode. • Allow a small amount of agar to solidify around the tip of a pH electrode in a beaker or cup. • Use a properly calibrated surface electrode.

The pH of liquid medium can also be taken after sterilization provided that it is allowed to cool to room temperature first.

To clean the pH meter, dip the tip into 50/50 isopropyl with water to remove all protein.

1NCCLS Volume 23 (M2–A8) January 2003

1.3.2 Dispensing Sterile Media Into Petri Dishes 1. Cool media to 50ºC. 2. Lay out sterile Petri dishes on a level surface. 3. Mix the media gently by rotating the flask. Avoid formation of air bubbles. 4. Flame and sterilize the mouth of the flask and pour required volume of media into each dish. This is dependent on size of dish. Depth should be not less than 5mm and should be even. 5. If air bubbles are formed while pouring, quickly pass the flame over the surface of the media before gelling occurs. The surface of the media should be smooth without bubbles. 6. Rotate the dish on the surface of the bench to ensure an even layer of agar. 7. When the media has cooled and gelled, label plates with name of media, and date of preparation. 8. Place plates in plastic bags to prevent loss of moisture and reduce risk of contamination. 9. Store in the refrigerator at 2–8ºC. 10. Do not issue for use until sterility testing and quality controls are done on the media.

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1.3.3 Dispensing Of Media Into Tubes 1. Place clean/sterile tubes in rack. 2. Dispense required volume of media aseptically into tubes. 3. Place caps on the tubes. Do not tighten. 4. Place autoclave indicator tapes across the tubes. 5. Autoclave at 121ºC for 12 to 15 minutes. 6. Place the rack in a slanted position and allow media to cool. (Solid or semi solid media). Make sure the required depth of the butt is achieved. For semi solid media without slant and liquid media leave in an upright position to cool.

Sugars should be sterilized by filtration. They cannot be autoclaved because they are unstable when heated.

1.3.4 Depth of Plates When pouring plates by hand pour plates at the depth of 4–5mm. Remove 10% of the total batch of plates poured and using a caliper measure the depth of the agar from the base of the Petri dish to the level of the agar.

Base of Petri Dish If media is poured using an automatic dispenser, the volume and depth of the media is measured initially. When the correct depth is achieved, the remainder of the plates is then poured.

2.0 Media Quality Control Methods 2.1 Introduction Culture media are vital to microbiology and good laboratory results are dependent on the use of quality media.

There are several stages culture media undergo before reaching the laboratory. a. Raw materials must be selected, tested or purchased to quality specifications. b. The manufacturer of these media is subjected to standing operating procedures by process controls and good manufacturing practices. c. Extreme importance is attached to labeling, the quality of the packaging and storage of their product. However, certain major characteristics do exist and must be established.

Extensive quality testing does not guarantee that a culture medium will be capable of recovering all strains of the desired organism; however, quality control results will give assurance of its ultimate performance.

2.2 Principles of QC of Media 1. Selective media should be tested with both organisms expected to grow and with organisms that should be inhibited. 2. New batches of media from new suppliers should be documented and validated for shelf life. 3. Reference cultures for tests on media should be extensive. 4. There must be continuous comparison of suppliers. 5. QC testing of the final product should not be the substitute for proper control of the processing of culture media.

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2.3 Shelf-life The shelf-life of prepared medium depends on: • The quality and formulation of the dehydrated culture medium. • The preparation process. • The storage conditions.

The shelf-life of the prepared medium should be assessed if it is intended to store the product before use. Shelf- life studies should be carried out using the standard process: a. Specific sterilization cycle b. Equipment c. Volume d. Water quality e. Vessels f. Storage conditions Subsequent batches of the same medium, prepared using the same standard process, can then be assigned a shelf-life, according to the results of the evaluation.

2.4 Standardization of Laboratory Media • Each medium should be included in the test programme. • Selection of standard is from a known, acceptable batch and set aside for future use. • Standard can be prepared according to the routine process and used as a comparator for the batch in use. • Use of a standard medium on specific or every occasion (for all media or only for specified media) at defined time or volume intervals, is the responsibility and decision of each laboratory. • Requirement for new standard is dependent on the use of the original batch or expiration of shelf-life. • Use of standard media is important to ascertain its importance and insurance of variations in inocula, viability or phenotypic characteristics are not compromised, but controlled for each test.

2.5 Control Medium • This medium is nutritious (sometimes differential) and is used to assess the number of colony-forming units (cfu). • Used also to assess the degree of inhibitions of a selective medium.

2.6 Test Programme Based on examination of physical characteristics and measurement of microbiological performance.

2.7 Physical Characteristics • Appearance of powder (homogenous or free-flowing powder) • Colour of product before and after sterilization • Clarity of product • Nature of gel • pH of product • Depth of medium (e.g. Mueller Hinton agar)

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2.8 Quality documentation • Conformance containing details of physical and microbiological tests carried out on a specific lot. • Documentation file comprising comprehensive information about: a. Manufacturer b. Accreditation/registration to EN ISO 9000 c. Product specification d. Labeling e. Storage f . Safety data

2.9 Methods Controlling the media preparation process.

2.9.1 Dust control: Use dried media in conditions appropriate to the hazard classification (Data supplied by manufacturer).

2.9.2 Weighing: Weigh powders accurately on a suitable balance. Balance should be routinely serviced and calibrated.

2.9.3 Manufacturer’s instructions: Follow instructions for preparation and use of culture media.

2.9.4 Heat processing • Culture media may deteriorate and lose performance during heat processing. Process is exacerbated during extended heating cycle. Chemo oxidation of culture media on exposure to heat gives rise to the production of peroxides and super oxides. • Plan and test heating cycles using thermocouples with specific volume and vessel combinations. • Ideal heating cycle is the minimum cycle which destroys all organisms. • Record heat processing cycles (temperature and time). • Retain data to allow full investigation of QC failure or reduced performance during shelf-life of prepared medium. • Equipment used in heat processing (i.e. autoclave and media preparators) should be calibrated and serviced as appropriate.

2.9.5 Batch rotation: Use batches of media in batch number order.

2.9.6 Record keeping • Keep adequate records of batches of medium received, processed, tested and used. • Undertake audit or investigation of batch failure, for which records are essential. • Equipment used for the preparation and QC testing should be routinely calibrated and serviced.

2.10 Examination of Physical Characteristics 2.10.1 Appearance of Powder Check appearance of powder for consistency, (homogenous or free-flowing).

2.10.2 Colour Compare the colour of the reconstituted and sterilized medium with that of the laboratory standard.

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2.10.3 Gel Test freshly poured surface-dried plates with an inoculation loop. The agar should be firm and workable, but not over-hard.

2.10.4 pH Measure pH of the medium in a beaker with suitable pH electrode. Gels may be tested either in a beaker or plate with a surface electrode.

General • Check prepared plates for cracked Petri dishes • Broken tubes or cracked tubes • Excessive bubbles after pouring plates and contamination • Haemolysis (Blood agar) • Cracked medium in plates • Excess volume

2.11 Test for Microbiological Performance • QC testing of media should be quantitative and qualitative analysis. • Growth, recovery rates and selectivity should be assessed together with colonial morphology, fermentation reactions, gas production and diagnostic reactions such as haemolysis, bile precipitation, lecithinase production and proteolysis. • Mixed cultures provide information on the natural competition of organisms and are particularly useful for testing of selective isolation media. • Pure cultures are convenient for routine testing sufficient to assess the performance of routine batches of media. • Mixed cultures are useful in the assessment of the overall effect of microbiological population, i.e. a. Type and numbers b. Ability of medium to grow c. Inhibition and differentiation between species

• Both mixed cultures and liquid cultures should be inoculated to a non-selective differential medium for assessing the time proportion of numbers. • Control of inoculum density is very important and inocula should be prepared by standard method: a. Overnight cultures b. Four-hour broth cultures or suspensions c. Use of turbidity standards

• Heavy inoculum produces atypical growth, atypical biochemical reactions and may prevent detection of deficiencies within the medium. • Inocula of inconsistent density makes assessment of selective media difficult. • Variation in inocula cause inconsistent results for inhibition and growth and make interpretation of routine QC results unreliable.

2.12 QC Tests for Solid Media 2.12.1 Streaking by standard method Care must be taken in streaking plates so that inoculum is transferred only from the preceding inoculation segment, to ensure adequate dilution resulting in single colonies

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2.12.2 Ecometric method A semi-quantitative method which involves the inoculation of organisms on to solid media in a standardized pattern to achieve ever-decreasing numbers of colony-forming units on the surface of the plate.

2.12.3 Miles and Misra technique Classic, quantitative surface plating technique, with a number of modifications based on a method originally described by Miles, Misra and Irwin.

2.12.4 Spiral plate method An automated inoculation system which has been used for counting micro organisms in food, milk and milk products and has been found to be equivalent to the standard pour plate procedure.

2.12.5 Pour plate technique Pour plate technique may be tested by performing a plate count and, where appropriate, a differential count. Dilutions of the test organisms or of a mixture of organisms are inoculated into a Petri dish and molten, cooled medium added.

2.12.6 Direct Inoculation Exercise care if using direct inoculation. An inoculum that is either too heavy or too light will mask both the growth and/or inhibitory properties of a medium. If a QC failure occurs while using direct inoculation, repeat using a standardized suspension as part of corrective action.

2.12.7 Standardized Suspension Using a standardized suspension of the QC organisms is a better challenge of media performance than direct inoculation. A standardized suspension also allows comparison of QC results between users. A more concentrated suspension tests the ability of selective media to successfully inhibit certain organisms and a lighter suspension challenges the ability of nonselective media to adequately support growth.

2.12.7.1 Preparation of suspension Prepare a suspension in sterile, nonbacteriostatic saline (0.85% a/v NaCl) to match a 0.54 McFarland standard (0.08 to 0.1 absorbance units at 625nm) (1x108 cfu/mL). Use an 18 to 24 hour culture of the QC organism.

2.12.7.2 Alternatively, prepare a suspension by inoculating 3 to 5 colonies from a 34 hour culture into sterile broth. Incubate for several hours to achieve a suspension equivalent to a 0.5 McFarland standard.

2.12.8 Dilution for Nonselective Media Dilute the suspension 1:100 in sterile broth or nonbacteriostatic saline. Inoculate each test plate with 10 μL (0.01 mL) of the suspension. Streak for isolation. If the 1:100 dilution inoculum proves too dense, use a 1:1000 dilution to produce isolated colonies.

2.12.9 Dilution of suspension for selective media Dilute the suspension 1:10 using sterile broth or nonbacteriostatic saline. Inoculate each test plate with 10 μL (0.01mL). streak for isolation. Use a 1:100 dilution if isolated colonies are not produced.

2.12.10 Tubed media Inoculate with 10 μL (0.01mL) of the undiluted 0.5 McFarland suspension.

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2.13 QC Tests for Liquid Media Assessment of the performance of liquid medium is more difficult than for solid medium. a. Modified Miles and Misra technique may be employed b. Selective liquid medium may require subculture to a solid medium after incubation c. Laboratories using liquid media should compile QC testing specifications and methods giving inoculum concentration and expected results

2.14 Interpretation of Microbiological Tests on Liquid Media • Equal growth assessed visually in both test and standard media that have been inoculated with the same number of organisms indicates satisfactory performance. • For selective media, quantitative subculture onto an appropriate solid medium enables critical comparison. • Employment of mixtures of organisms, subculture should be made onto a medium that will reveal all test organisms, since a selective medium (e.g. DCA) will obscure the true mixture. • Growth should be assessed with reference top the original inoculum (colony-forming units).

2.15 Sterility Tests • Incubate samples from a batch for two to five days at 30ºC and/or 37ºC. (A lot of 100 units or less, 3–5% sample should be tested.) • For a large batch, select 10 plates or tubes randomly. • There should be no evidence of microbial growth after incubation. • Discard all sterility samples after tests have been completed.

2.16 Quality Control Limits for Haemophilus Test Medium Agar

Antimicrobial Agent Disc Content Quality Control Quality Control Range (mm) Range (mm) Haemophilus influenzae ATCC 49247 ATCC 49766 Amoxicillin/Clavulanic Acid 20/10μg 15–23 – Ampicillin 10μg 13–21 – Ampicillin - Sulbactam 10/10μg 14–22 – Aztreonam 30μg 30–38 – Cefaclor 30μ – 25–31 Cefamandole 30μ – 29–37 Cefixime 5μg 25–33 – Cefonicid 30μg – 30–38 Cefotaxime 30μg 31–39 – Cefazidime 30μg 27–35 – Ceftizoxime 30μg 29–39 – Ceftriaxone 30μg 31–39 – Cefuroxime 30μg – 28–36 Chloramphenicol 30μg 31–40 – Ciprofloxacin 5μg 34–42 – Imipenem 10μg 21–29 – Rifampicin 5μg 22–30 – Tetracycline 30μg 14–22 – Trimethoprim/ 1.25/23.75μg 24–32 – Sulfamethoxazole

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2.17 Storage of Culture Media 2.17.1 Dehydrated Product When storing the dehydrated product, the manufactures recommendations should be followed. The normal temperature for storage of the dehydrated product is 2–25ºC. Some media may have special temperatures. This is stated on the packaging.

2.17.2 Prepared Media Prepared media in plates should be quarantined during quality control testing. Once this process is completed, media can then be placed into general circulation. Prepared culture media should be stored at 4–8ºC under normal conditions unless other wise stated.

Media in Petri dishes should be stored in the original Petri dish sleeve wrapping. Media containing dyes must be protected from sources of light.

During storage, media showing any evidence of contamination, discolouration, drying, cracking or other signs of deterioration. Tubes of sterile fermentation media should be handled carefully to avoid entrapping air in the inner fermentation vials. Prepared broth media should be stores in screw cap tubes or other tightly sealed tubes.

2.17.3 Quality Control Data Records See Appendices 3 and 4 located at the back of this procedure.

3.0 Quality Control of Culture Media Prepared In-house 3.1 Procedure • All prepared media must be examined visually for colour change, precipitation, lysis of blood, contamination, gelling etc. • Report any atypical observation to the QA technologist or manager. • Incidents form to be documented. • Perform pH testing after solidified and cooled to RT. Record value obtained. • Perform QC testing on all media.

3.2 Quality Control Protocols Quality control testing is required for all laboratory-prepared media and for some kinds of commercially prepared, ready-to-use media. Work sheets should be prepared for each lot of media used in the laboratory. The work for each lot of medium should be retained in file for a minimum of 2 years.

The following procedures for laboratory-prepared media must be observed: 1. General procedures and formulations should be available for each medium prepared. 2. Dehydrated media received from commercial manufacturers should be dated on receipt and again when opened. 3. The manufacturer’s expiration date should be noted. 4. The amount, source, lot number and method of sterilizing all components, the date of preparation, the expiration date, the quantity prepared and the initials of the preparer should be carefully recorded. 5. Autoclave temperature-recording charts should be saved and monitored, since excessive heating is a common source of error. 6. A representative sample of each lot of laboratory-prepared media should then be sterility tested. 7. Criteria for random selection for sterility must be standardized. 8. Bacteriologic media are incubated for 48 hrs and media for mycology and mycobacteriology are incubated for 5 days. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN 17 A CARIFORUM Project Funded by the European Union and Implemented by CAREC Media Preparation and Quality Control

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9. If more than 10% of plates or tubes are contaminated, the entire lot of medium must be incubated at 35°C for 24 hrs. If more than 3% of the entire lot is contaminated, the media should be discarded. 10. If less than 3% of the entire lot is contaminated, the contaminated plates or tubes should be discarded and the expiration date should be changed to one half of the usual dating, for example, 4 weeks instead of 8 for plates sealed in bags and 3 months instead of 6 for screw-capped, tubed media. 11. Blood and other supplements are sterility tested before use. 12. Each lot of medium must be tested to determine if it is suitable for laboratory use. Samples of commercial or laboratory-prepared media should be inoculated with stock organisms of known physiologic and biochemical properties. 13. Reference organisms should demonstrate the selectivity, inhibitory effects, colonial morphology and growth characteristics or biochemical reactions for which the medium is intended. 14. Media that fail to perform satisfactorily after repeat testing must be discarded. 15. The CLSI (formerly NCCLS) standard recommends that some commercially prepared, ready-to-use media need not be retested by purchasers, provided that the media are obtained from commercial sources that employ the quality control criteria recommended by CLSI (formerly NCCLS) and assure the purchaser that the criteria have been met. Please refer to Section 4: Quality Control of Commercially Prepared Media, and Appendix 5.

3.3 Thymidine/Thymine Levels • This test is used to evaluate new lots of Mueller Hinton agar. • Media containing excessive amounts of thymidine or thymine can reverse the inhibitory effect of sulfonamides and trimethoprim, thus yielding smaller and less distinct zones, or even no zone at all, which may result in false-resistance reports. • An antimicrobial susceptibility test is performed using faecalis ATCC 29212 or Enterococcus faecalis ATCC 33186 with trimethoprim-sulfamethoxazole disks. • Satisfactory media will provide essentially clear, distinct zones of inhibition 20mm or greater in diameter • Unsatisfactory media will produce no zone of inhibition, growth within the zone, or a zone of less than 20mm.

4.0 Quality Control of Commercially Prepared Media 4.1 Exempt Media Category This category includes commercial media documented to maintain consistent user performance with minimum variation and requires minimum quality control (Appendix 5).

Laboratories are advised to carefully monitor the performance of media used for fastidious organisms or specialty testing, regardless of categorization of exempt, in order to ensure that recovery of isolates from clinical specimens is satisfactory. Categorization of media as exempt does not preclude a laboratory from performing complete quality control on any manufactured medium type when deemed necessary.

4.2 Nonexempt Media Category. This category includes commercial media documented by user quality control performance data collected by CAP to vary in performance from lot to lot, or any media prepared by the user. Nonexempt media requires complete user QC including confirmation of satisfactory performance with recommended organisms (Appendix 2).

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4.3 General QC Requirements for both Exempt and Nonexempt Media. 4.3.1 Receipt of Media from Manufacturer The following procedures should be performed upon receipt: a. Verify delivery of the ordered amount. Check each medium type for multiple lot numbers and/or impending expiry dates. Report recurring problems to manufacturer or distributor. b. Record the lot number, expiration date, and received date for each medium type. c. Store the media as specified by the manufacturer pending QC.

4.3.2 Visual Inspection Record the numbers of plates per lot that exhibit any of the characteristics listed below. If excessive, notify the manufacturer or distributor.

Visually inspect each medium lot for the following: • cracked or damaged plates • agar detached from the petri-dishes • frozen or melted agar • unequal filling of plates • insufficient agar in the plates (<3 mm) • haemolysis of blood-containing media • change in the expected colour of the media\excessive bubbles or rough surfaces • excessive moisture or dehydration • obvious contamination • presence of precipitates

4.3.3 Checking for Contamination Because contamination testing is routinely performed by manufacturers, commercially prepared media need not be re-tested for sterility by the end user. Instead, careful inspection for contamination should take place immediately before inoculation with patient specimens.

4.3.4 Quality Assurance Observations have confirmed that both exempt and nonexempt media have the potential to perform less than optimally or may fail QC after receipt from the manufacturer. In order to monitor for these potential failures, users are encouraged to adopt the following procedures: a. Laboratories must use media for primary culture which will support the growth of a wide variety of organisms, e.g. blood agar and chocolate agar. b. Laboratories are strongly urged to confirm the ability of the ability of media to support the growth of fastidious organisms.

4.4 Q.C. Requirements for Nonexempt Media Only Nonexempt media require performance of complete user QC. Perform visual and contamination checks. In addition, verify acceptable growth and/or inhibitory properties with appropriate bacterial or fungal control organisms.

4.5 Reporting Deficiencies Identified by the User a. Identify and/or correct the cause of any media failure. Document all activities b. Notify the manufacturer and/or distributor. Document the manufacturer’s/distributor’s response in corrective action.

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5.0 Quality Control of Media not included in Section 6

No Medium Control Organisms ATCC Incubation Conditions Expected Results

Mueller Hinton 35–37ºC, CO , 1 E. coli 25922 2 Good to luxuriant with blood 18–24 hrs

S. aureus 25923 As above As above

Streptococcus 6305 As above Good growth pneumoniae

Good to luxuriant 35–37ºC, aerobic, 2 Selenite Broth typhimurium 14028 colourless when subculture 18–24hrs to MacConkey

No increase in numbers E. coli 25922 As above white to pink when subculture to MacConkey agar

35–37ºC aerobic, Reaction positive 3 Dextrose Agar E.coli 25922 18–24hrs yellow colour change

Salmonella typhimurium 14028 As above As above

Reaction positive 4 Maltose E.coli 25922 As above yellow colour change

Reaction negative dysenteriae 13313 As above no colour change

Reaction positive 5 Lactose E.coli 25922 As above yellow colour change

Reaction negative Salmonella typhimurium 14028 As above no colour change

Reaction positive – yellow 6 Sucrose Agar Klebsiella 13883 As above colour change

Negative reaction Shigella sonnei 25931 As above no colour change

Positive reaction 7 D-Mannitol Agar S. aureus 25923 As above colour change to yellow

Negative reaction Pseudomonus aeruginosa 27853 As above no colour change

8 Motility test Medium E.coli 25922 As above Reaction positive – growth

Enterobacter aerogenes 13048 As above Reaction positive – growth

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No Medium Control Organisms ATCC Incubation Conditions Expected Results

Reaction negative – Klebsiella pneumoniae 13883 As above no growth Reaction negative – Staph aureus 25923 As above no growth Reaction negative – 9 Malonate Broth E.coli 25922 As above no change in colour Reaction positive – colour Klebsiella pneumoniae 13883 As above change from green to blue

Enterobacter aerogenes 13048 As above As above

Haemophilus 35–37ºC, CO , 10 S. aureus 29213 2 No growth Isolation Agar 18–24 hrs

H. Influenzae 102211 As above Growth

35–42ºC, CO , 11 Campylobacter Agar Campylobacter jejuni 29428 2 Growth 24–48 hours

E.coli 25922 As above No growth MacConkey Agar 35–37ºC, aerobic, Luxuriant growth, 12 without salt E.coli 25922 18–24 hrs pink to red colonies (OXMAC) Luxuriant growth, Salmonella enteritidis 13076 As above colourless colonies Good growth, colourless Shigella flexneri 12022 As above colonies Luxuriant growth, slightly Enterobacter aerogenes 13048 As above pink to pink colonies S. aureus 25293 As above Good growth, pink colonies

Good to excellent growth, Eosin Methylene 35–37ºC aerobic, colonies purple with black 13 E. coli 25922 Blue Agar 18–24 hrs centre and green metallic sheen

Good growth, Enterobacter aerogenes 13048 As above colonies pink, no sheen

Good growth, colonies Klebsiella pneumoniae 13883 As above with green metallic sheen, dark centres

Good to excellent growth, Proteus mirabilis 25933 As above colourless colonies

None to poor growth, S. aureus 25923 As above colourless colonies

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No Medium Control Organisms ATCC Incubation Conditions Expected Results

Good to excellent growth, Candida albicans 10231 As above colourless colonies Streptococcus 14 Veal Infusion Agar 6305 As above Luxuriant growth pneumoniae

S. epidermidis 12228 As above Luxuriant growth

Luxuriant growth 15 Skim Milk E. coli 259222 As above Proteolytic activity –

Luxuriant growth Bacillus subtilis 6633 As above Proteolytic activity +

Pseudomonas Luxuriant growth 27853 As above aeruginosa Proteolytic activity +

Luxuriant growth Proteus mirabilis 25933 As above Proteolytic activity +

Luxuriant growth Enterococcus faecalis 29212 As above Proteolytic activity -

Viable turbidity; good growth on subculture to Streptococcus Blood agar at 35°C: 16 bottles 6305 35°C, vented for 7 days pneumoniae gram stain of uninoculated (aerobic) medium should show no Visible turbidity; good growth on subculture to Blood culture anaerobic blood agar at 17 bottles Bacteroides fragilis 25285 35°C, unvented, 7 days 35°C in anaerobic (anaerobic) atmosphere; Gram stain of uninoculated media should show no bacteria

Good growth on agar and Blood culture in broth after 48 hrs; Gram 18 bottles-biphasic Candida albicans 10231 30°C vented, 7 days stain of uninoculated media for fungus should show no bacteria or fungi

Inoculum – 100–1000 Candida BCG 30+/- 2°C, 19 Candida albicans 10231 Growth – good Agar base 24-48 hrs Colour of medium – yellow

Inoculum – 1000-2000 E.coli 25922 As above Growth – inhibited Colour of medium – green

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No Medium Control Organisms ATCC Incubation Conditions Expected Results

CCDA Campylobacter 37–42°C for 20 Campylobacter jejuni 29428 Growth – luxuriant Blood-free 18–48 hrs Selective Medium

E.coli 25922 As above Growth – poor to inhibited

Invert plates within 15 Zone sizes should fall minutes after discs are within ranges specified in Haemophilus applied and incubate 21 Haemophilus influenzae 49247 Tables 1 and 2 for the Testing Medium 35+/–2°C in an aerobic appropriate organism/drug atmosphere enriched with combinations carbon dioxide

Haemophilus influenzae 49766 As above Growth of ATCC 10211 Haemophilus influenzae 10211 As above. should be moderate to heavy 25–30°C in aerobic Sabouraud 22 Candida albicans 60193 atmosphere up to Growth Dextrose Agar 7 days Trichophyton 9533 As above Growth mentagrophytes

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6.0 Methods 6.1 Preparation of Alkaline Peptone Water 6.1.1 Purpose To ensure that the correct validated procedure is followed for the preparation of alkaline peptone water in order to provide accurate, reliable and reproducible results having clinical utility.

6.1.2 Introduction Alkaline peptone water is an enrichment medium used for the cultivation of Vibrio species from faeces and other infected materials. Clinical materials containing small numbers of Vibrio should be inoculated into an enrichment medium prior to plating onto a selective medium, such as TCBS Agar. Alkaline Peptone Water is a suitable enrichment broth for this purpose. The relatively high pH of the medium (approximately 8.4) provides a favorable environment for the growth of vibrios. Enzymatic digest of casein provides amino acids and other complex nitrogenous substances necessary to support bacterial growth. Sodium chloride maintains osmotic equilibrium.

6.1.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of alkaline peptone water as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.1.4 Staff Competence Trained qualified competent staff

6.1.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.1.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.1.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Alkaline peptone water base Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Glass tubes Incubator Spirit Lamp pH meter/paper

6.1.8 Procedure • Prepare according to manufacturer’s instructions • Dispense into appropriate glassware • Perform QC and sterility check • Store at 2–8ºC

6.1.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

6.1.10 Limitations and Pitfalls This prepared tube medium is intended to be used as an enrichment medium. A pure culture is recommend- ed for biochemical tests and other identification procedures.

6.1.10 References 1. Difco Manual 11th edition. 1998. Difco Laboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.2 Preparation of Blood Agar 6.2.1 Purpose To ensure that the correct validated procedure is followed for the preparation of blood agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.2.2 Introduction Blood Agar Base (Infusion Agar) and Blood Agar Base No. 2, with the addition of sterile blood, is used for the isolation, cultivation and detection of hemolytic activity of streptococci and other fastidious microorganisms. Infusion agar is an all-purpose medium which has been used for many years as a base for the preparation of blood agars. In a study of viability of streptococci, Snavely and Brahier performed comparative studies of horse, rabbit and sheep blood with Blood Agar Base, and found that sheep blood gave the clearest and most reliable colony and haemolysis characteristics at both 24 and 48 hours. In the course of the investigation, about 1,300 isolations of streptococci were made with Blood Agar Base containing 5% sheep blood. Blood Agar Base No. 2 is a nutritionally rich medium for maximum recovery of fastidious microorganisms. Blood Agar Base media are specified in standard methods for food testing. 2–4 Infusion Agar and Blood Agar Base No. 2 have been largely replaced as blood agar bases by the Tryptic/Trypticase™ Soy Agar formulations, which contain milk and plant peptones in place of the variable infusion component.

6.2.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of blood agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.2.4 Staff Competence Trained competent personnel.

6.2.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.2.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.2.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Blood agar base N/A Sheep blood Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp pH meter/paper

6.2.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into sterile Petri dishes 3. Test samples of the finished product for performance using stable, typical control cultures. Perform sterility check. 4. Label and store media at 2–8ºC

6.2.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Staph aureus 25923 35–37ºC, aerobic, 18–24 hours Growth +

Streptococcus pyogenes 19615 35–37ºC, aerobic, 18–24 hours Growth + Beta haemolysis

Streptococcus pneumoniae 6303 35–37ºC, aerobic, 18–24 hours Growth + Alpha haemolysis

E. coli 25922 35–37ºC, aerobic, 18–24 hours Growth +

Haemophilus influenzae 19418 35–37ºC, aerobic, 18–24 hours Growth + Growth + N. meningitidis 13090 35–37ºC, aerobic, 18–24 hours Gamma haemolysis 27853 35–37ºC, aerobic, 18–24 hours Growth +

6.2.10 Limitations and Pitfalls Colonies of Haemophilus haemolyticus are beta-hemolytic on horse and rabbit blood agar and must be distinguished from colonies of beta-hemolytic streptococci using other criteria. The use of sheep blood has been suggested to obviate this problem since sheep blood is deficient in pyridine nucleotides and does not support growth of H. haemolyticus.

6.2.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.3 Preparation of Bile Esculin Agar 6.3.1 Purpose To ensure that the correct validated procedure is followed for the preparation of bile esculin agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.3.2 Introduction Bile esculin agar base (with added esculin) and bile esculin agar are differential media used for isolating and identifying group D streptococci. The ability to hydrolyze esculin in the presence of bile is a characteristic of enterococci and group D streptococci. Swan compared the use of an esculin medium containing 40% bile salts with the Lancefield serological method of grouping. He reported that a positive reaction on the bile esculin medium correlated with a serological Group D precipitin reaction. Facklam and Moody in a comparative study of tests used the presumptively identify group D streptococci, found that the bile esculin test provided a reliable means of identifying group D streptococci and differentiating them from non-group D streptococci. Facklam further confirmed the usefulness of bile esculin agar in another study differentiating enterococci / group D streptococci from non-group D streptococci.

6.3.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of bile esculin agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.3.4 Staff Competence Trained qualified competent staff

6.3.5 Safety Instructions Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable gloves and eye/face protection. Use only in well ventilated areas. Keep container tightly closed.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.3.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.3.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Bile esculin agar base Esculin Distilled Water Bile esculin agar Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes/glass tubes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.3.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into sterile Petri dishes 3. Test samples of the finished product for performance using stable, typical control cultures. Perform sterility check. 4. Label and store media at 2–8ºC

6.3.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + Enterococcus faecalis 29212 35–37ºC, aerobic, 18–24 hours Hydrolysis +

E. coli 25922 35–37ºC, aerobic, 18–24 hours Complete inhibition

Partial to complete inhibition Streptococcus pyogenes 19615 35–37ºC, aerobic, 18–24 hours Hydrolysis –

6.3.10 Limitations and Pitfalls 1. Strains of Lactococcus, Leuconostoc and Pediococcus that give a positive bile-esculin reaction have been isolated from human infections. 2. Occasional strains of viridans streptococci blacken the medium or display weakly positive reactions.

6.3.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.4 Preparation of Baird Parker Agar 6.4.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Baird Parker agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.4.2 Introduction Studies done by Tardio and Baer and Baer indicated that the Baird Parker media is more suitable for the isolation and enumeration of S. aureus than the Vogel and Johnson agar.In this study, it was shown that Baird- Parker Agar was less inhibitory than Vogel and Johnson Agar for selected strains of S. aureus and that it possesses a diagnostic aid (egg yolk reaction) not present in Vogel and Johnson Agar. Baird–Parker Agar Base contains peptone, beef extract and yeast extract as sources of nitrogenous compounds, carbon, sulphur, vitamins and trace minerals. Sodium pyruvate is incorporated in order to stimulate the growth of S. aureus without destroying the selectivity. The tellurite additive is toxic to egg yolk clearing strains other than S. aureus and imparts a black colour to the colonies. The egg yolk additive, in addition to being an enrichment, aids in the identification process by demonstrating lecithinase activity (egg yolk reaction). Glycine and lithium chloride have inhibitory action for organisms other than S. aureus.

6.4.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Baird Parker agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.4.4 Staff Competence Trained qualified competent staff

6.4.5 Safety Instructions Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. May cause harm to the unborn child. Wear suitable gloves and eye/face protection. Keep container tightly closed. TARGET ORGANS: Blood, kidneys and nerves.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

5.4.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.4.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Baird Parker Agar Base N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes/glass tubes Incubator Spirit Lamp pH meter/paper

6.4.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into sterile Petri dishes 3. Test samples of the finished product for performance using stable, typical control cultures. Perform sterility check. 4. Label and store media at 2–8ºC

6.4.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + Colony colour: Staph aureus 25923 35–37ºC, aerobic, 24–48 hours Grey/blask, shiny Lecithinase +

Growth + Colony colour: Proteus mirabilis 25933 35–37ºC, aerobic, 24–48 hours brown-black Lecithinase -

Growth + Staph epidermidis 12228 35–37ºC, aerobic, 24–48 hours Colony colour: black Lecithinase -

6.4.10 Limitations and Pitfalls Baird-Parker is selective for coagulase-positive staphylococci, but other bacteria may grow. Microscopic examination and biochemical tests will differentiate coagulase-positive staphylococci from other organisms.

6.4.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.5 Preparation of Bile Esculin Azide Agar 6.5.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Bile Esculin Azide agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.5.2 Introduction Bile Esculin Azide agar is used for isolating, differentiating and presumptively identifying group D streptococci. Organisms positive for esculin hydrolysis hydrolyse the glycoside esculin to esculetin and dextrose. Esculatin reacts with ferric ammonium citrate to form a dark brown or black complex. Oxgall (bile) inhibits gram-positive bacteria other than enterocci while sodium azide inhibits gram-negative bacteria. Tryptone and peptone No. 3 provide nitrogen, vitamins and minerals. Yeast extract provides vitamins and cofactors required for growth, as well as additional sources of nitrogen and carbon. Sodium Chloride maintains the osmotic balance of the medium. Bacto agar is the solidifying agent.

6.5.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Bile Esculin Azide agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.5.4 Staff Competence Trained qualified competent staff

6.5.5 Safety Instructions Irritating to eyes, respiratory system and skin. Harmful by inhalation and if swallowed. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container tightly closed. TARGET ORGANS: Cardiovascular, lung and nerves.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.5.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.5.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Bile Esculin Azide Agar Horse serum (optional) Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes/glass tubes Incubator Spirit Lamp pH meter/paper

6.5.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into sterile Petri dishes 3. Test samples of the finished product for performance using stable, typical control cultures. Perform sterility check. 4. Label and store media at 2–8ºC

6.5.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Growth + Enterococcus faecalis 29212 18–24 hours Hydrolysis + 35–37ºC, aerobic, Growth + Proteus mirabilis 25933 18–24 hours Hydrolysis - 35–37ºC, aerobic, Growth + Staph aureus 25923 18–24 hours Hydrolysis - 35–37ºC, aerobic, Growth: none to poor Streptococcus pyogenes 19615 . 18–24 hours Hydrolysis - Hydrolysis + = blackening of the media

6.5.10 Limitations and Pitfalls 1. S. aureus and S. epidermidis may exhibit growth on the medium but will show no action on the esculin. 2. Other than the enterococci, L. monocytogenes consistently blackens the medium around colonies. After 18–24hrs there may be a reddish to black brown zone of hydrolysis surrounding pinpoint listeria colonies. After 48hrs white grey pigmented colonies will be seen. Listeria do not attain the same degree of esculin hydrolysis displayed by enterococci in this short incubation period.

6.5.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.6 Preparation of Chocolate Agar 6.6.1 Purpose To ensure that the correct validated procedure is followed for the preparation of chocolate agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.6.2 Introduction Chocolate II Agar is an improved medium for use in qualitative procedures for the isolation and cultivation of fastidious microorganisms, especially Neisseria and Haemophilus species, from a variety of clinical specimens. Chocolate II Agar with Bacitracin is a selective medium used for the isolation of Haemophilus species. Chocolate II Agar with Pyridoxal is used for the isolation of nutritionally-variant streptococci (vitamin B6- requiring streptococci) from blood cultures. In a study done by Carpenter and Morton it was shown that chocolate medium supplemented with yeast concentrate aided better growth of gonococci and other fastidious organisms e.g. Haemophilus. The yeast concentrate has been replaced by an enrichment supplement e.g. BBL™ IsoVitaleX™ and Oxoid VitoxTM. Chocolate II Agar is often used as the medium for subculture from blood culture bottles to detect the presence of bacteria in cases of septicaemia. Some cases of septicaemia are caused by organisms referred to as ‘nutritionally variant streptococci’. Chocolate II Agar contains an improved GC Agar base (GC II Agar Base), bovine hemoglobin and enrichment supplement. GC II Agar Base contains nitrogenous nutrients in the form of casein and meat peptones, phosphate buffer to maintain pH and corn starch, which neutralizes toxic fatty acids that may be present in the agar. Hemoglobin provides X factor (hemin) for Haemophilus species. The enrichment supplement is a defined supplement which provides V factor (nicotinamide adenine dinucleotide, NAD) for Haemophilus species and vitamins, amino acids, co-enzymes, dextrose, ferric ion and other factors which improve the growth of pathogenic Neisseria. Chocolate II Agar with Bacitracin is a selective medium for the isolation of Haemophilus species from specimens containing mixed flora. Bacitracin is a polypeptide antibiotic that inhibits gram-positive bacteria and Neisseria.

5.6.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of chocolate agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.6.4 Staff Competence Trained qualified competent staff

6.6.5 Safety Instructions Irritating to eyes, respiratory system and skin. Harmful by inhalation and if swallowed. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container tightly closed. TARGET ORGANS: Cardiovascular, lung and nerves.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.6.6 Pre-examination Procedures 1. Storage: Store GC and Haemoglobin dehydrated medium below 30ºC. Store Haemoglobin 2% at 15–30ºC. Store supplements at 2–8ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.6.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT GC Agar Haemoglobin/Haemoglobin 2% Distilled Water Supplement B or VX Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes/glass tubes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.6.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in Petri dishes 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.6.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Haemophilus influenzae 19418 35–37ºC, aerobic, 18–24 hours Growth +

Streptococcus pneumoniae 6303 35–37ºC, aerobic, 18–24 hours Growth +

Streptococcus pyogenes 19615 35–37ºC, aerobic, 18–24 hours Growth +

6.6.10 Limitations and Pitfalls 1. Chocolate agar is an enriched media on which pathogenic bacteria may be overgrown with undesirable or nonpathogenic bacteria.

6.6.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003. 3. BBL Quality Control and Product Information Manual for Plated Media. 1994. Becton Dickinson Microbiology Systems.

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6.7 Preparation of CLED Agar 6.7.1 Purpose To ensure that the correct validated procedure is followed for the preparation of CLED agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.7.2 Introduction In 1960, Sandys reported on the development of a new method of preventing the swarming of Proteus on solid media by restricting the electrolytes in the culture medium. This electrolyte-deficient medium of Sandys was modified by Mackey and Sandys for use in urine culture by substituting lactose and sucrose for the mannitol and increasing the concentrations of the bromthymol blue indicator and of the agar. These two investigators further modified the medium by the incorporation of cystine in order to enhance the growth of cystine-dependent “dwarf colony” coliforms and by deletion of sucrose. They designated the new medium as Cystine-Lactose-Electrolyte- Deficient (CLED) medium and reported it to be ideal for dip-inoculum techniques and for urinary bacteriology in general. The nutrients in CLED Agar are supplied by peptones, pancreatic digests of gelatin and casein, and beef extract. Lactose is included to provide an energy source for organisms capable of utilizing it by a fermentative mechanism. The cystine permits the growth of ‘dwarf colony’ coliforms. Bromthymol blue is used as a pH indicator to differentiate lactose fermenters from lactose nonfermenters. Organisms that ferment lactose will lower the pH and change the colour of the medium from green to yellow. Electrolyte sources are reduced in order to restrict the swarming of Proteus species.

6.7.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of CLED agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.7.4 Staff Competence Trained qualified competent staff

6.7.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.7.6 Pre-examination Procedures 1. Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.7.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT CLED Agar Horse serum (optional) Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes/glass tubes Incubator Spirit Lamp pH meter/paper

6.7.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in Petri dishes 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.7.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Growth + E. coli 25922 18–24 hours Lactose fermentation 35–37ºC, aerobic, Staph aureus 25923 Growth + 18–24 hours 35–37ºC, aerobic, Growth + Proteus vulgaris 13315 18–24 hours No lactose fermentation 35–37ºC, aerobic, Enterococcus faecalis 29212 Growth + 18–24 hours 35–37ºC, aerobic, Growth + Kleb. pneumoniae 13883 18–24 hours Lactose fermentation

6.7.10 Limitations and Pitfalls 1. Factors that may cause urine counts from infected patients to be low include: rapid rate of urine flow, prior initiation of antimicrobial therapy, a urine pH of less than 5 and a specific gravity of less than 1.003. 2. Since the nutritional requirements of organisms vary, some strains may be encountered that fail to grow or grow poorly on this medium. 3. CLED Agar is basically non-selective. However, due to the electrolyte exclusion, the growth of Shigella species is usually inhibited.

6.7.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.8 Preparation of Cooked Meat Medium 6.8.1 Purpose To ensure that the correct validated procedure is followed for the preparation of cooked meat medium in order to provide accurate, reliable and reproducible results having clinical utility.

6.8.2 Introduction Cooked Meat Medium is used for the cultivation of anaerobes, especially pathogenic clostridia. In 1916, Robertson developed a cooked meat medium for use in the cultivation of certain anaerobes isolated from wounds. The present formulation for Cooked Meat Medium is a modification of Robertson’s original formula. Cooked Meat Medium is still widely used for the cultivation and maintenance of clostridia and for determining proteolytic activity of anaerobes. For example, the medium is recommended for use in the enumeration and identification of perfringens from food. It supports the growth of most sporeforming and nonsporeforming obligate anaerobes and may be used for a variety of purposes including the maintenance of stock cultures. The medium is also useful as an enrichment broth for cultivating anaerobes that may be present in small numbers in a population and as a subculture medium for determination of proteolysis (meat digestion) and spore formation by Clostridium species. Cooked Meat Medium provides a favorable environment for the growth of anaerobes, since the muscle protein in the heart tissue granules is a source of amino acids and other nutrients.The muscle tissue also provides reducing substances, particularly glutathione, which permits the growth of strict anaerobes. The sulfhydryl groups, which exert the reducing effect, are more available in denatured protein; therefore, the meat particles are cooked for use in the medium. Sodium Chloride maintains the osmotic balance of the medium. The low concentration of dextrose is sufficient as the energy source but not high enough to accumulate toxic metabolites.

6.8.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of cooked meat medium as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.8.4 Staff Competence Trained qualified competent staff

6.8.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.8.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.8.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Cooked Meat Medium N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware with covers Incubator Spirit Lamp pH meter/paper

6.8.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.8.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, 12924 Growth + 40–48 hours 35–37ºC, aerobic, Bacteroides vulgatus 8482 Growth + 40–48 hours 35–37ºC, aerobic, S. aureus 25923 Growth + 40–48 hours 35–37ºC, aerobic, Enterococcus faecalis 29212 Growth + 40–48 hours

6.8.10 Limitations and Pitfalls Since the nutritional requirements of organisms vary some strains may be encountered that fail to grow or grow poorly on this medium.

6.8.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.9 Preparation of Brain Heart Infusion Broth 6.9.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Brain Heart Infusion Broth in order to provide accurate, reliable and reproducible results having clinical utility.

6.9.2 Introduction Brain Heart Infusion (BHI) is a general-purpose liquid medium used in the cultivation of fastidious and nonfastidious microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical and nonclinical materials. A supplemented pre-reduced formulation in tubes is especially recommended for the cultivation of anaerobes. BHI broth is used for the cultivation of a wide variety of microorganisms, including bacteria, yeasts and molds. BHI Broth is a nutritious, buffered culture medium that contains infusions of brain and heart tissue and peptones to supply protein and other nutrients necessary to support the growth of fastidious and nonfastidious microorganisms.

6.9.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of brain heart infusion broth as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.9.4 Staff Competence Trained qualified competent staff

6.9.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.9.6 Pre-examination Procedures 7.1 Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.9.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Brain heart infusion Vancomycin Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile tubes with covers Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.9.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.9.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Streptococcus 35–37ºC, aerobic, 6303 Growth + pneumoniae 18–24 hours 35–37ºC, aerobic, Streptococcus pyogenes 19615 Growth + 18–24 hours

6.9.10 Limitations and Pitfalls N/A

6.9.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.10 Preparation of Brain Heart Infusion Agar 6.10.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Brain Heart Infusion Agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.10.2 Introduction Brain Heart Infusion (BHI) is a general-purpose liquid medium used in the cultivation of fastidious and nonfastidious microorganisms, including aerobic and anaerobic bacteria, from a variety of clinical and nonclinical materials. A supplemented pre-reduced formulation in tubes is especially recommended for the cultivation of anaerobes. BHI broth is used for the cultivation of a wide variety of microorganisms, including bacteria, yeasts and molds. BHI Broth is a nutritious, buffered culture medium that contains infusions of brain and heart tissue and peptones to supply protein and other nutrients necessary to support the growth of fastidious and nonfastidious microorganisms. Agar is added as a solidifying agent. BHI agar may be supplemented with 6mg/L vancomycin to be used as a screen plate to presumptively identify vancomycin resistant enterococci (VRE).

6.10.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of brain heart infusion agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.10.4 Staff Competence Trained qualified competent staff

6.10.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.10.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.10.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Brain heart infusion Vancomycin Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile tubes with covers Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.10.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.10.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Streptococcus 35–37ºC, aerobic, 6303 Growth + pneumoniae 18–24 hours 35–37ºC, aerobic, Streptococcus pyogenes 19615 Growth + 18–24 hours

6.10.10 Limitations and Pitfalls N/A

6.10.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.11 Preparation of GN Broth 6.11.1 Purpose To ensure that the correct validated procedure is followed for the preparation of GN broth in order to provide accurate, reliable and reproducible results having clinical utility.

6.11.2 Introduction GN Broth is used for the selective enrichment of Salmonella and Shigella. GN (Gram Negative) Broth was developed by Hajna as an enrichment medium for the recovery of Salmonella and Shigella from clinical and nonclinical specimens. Croft and Miller succeeded in isolating more Shigella strains by use of this medium, rather than by direct streaking. Taylor and Schelhart reported that GN Broth enhanced the isolation of enteric pathogens, producing a 53% increase in Shigella and a 36% increase in Salmonella as compared to direct streaking.In another study, Taylor and Schelhart showed that GN Broth was superior to selenite enrichment media for the isolation of Shigella. Peptones provide amino acids and other nitrogenous substances to support bacterial growth. Mannitol and dextrose are sources of energy. Mannitol is provided in a higher concentration than dextrose to enhance the growth of mannitol-fermenting species, such as Salmonella and Shigella, and limit the growth of Proteus and other dextrose-fermenting bacteria. Sodium Citrate and Sodium Desoxychoate inhibit growth of gram-positive bacteria and of coliforms other than Salmonella and Shigella. Dipotassium Phosphate and Monopotassium Phosphate buffer the medium.

6.11.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of GN broth as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.11.4 Staff Competence Trained qualified competent staff

6.11.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.11.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.11.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT GN Broth N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile tubes with covers Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.11.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.11.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth in GN Broth: + Salmonella typhimurium 14028 37ºC, aerobic, 18–24 hours Growth when subcultured onto MacConkey: Good Growth in GN Broth: + Shigella flexneri 12022 37ºC, aerobic, 18–24 hours Growth when subcultured onto MacConkey: Good Growth in GN Broth: + E. coli 25922 37ºC, aerobic, 18–24 hours Growth when subcultured onto MacConkey: Good Growth in GN Broth: None to poor Enterococcus faecalis 29212 37ºC, aerobic, 18–24 hours Growth when subcultured onto MacConkey: None to poor 6.11.10 Limitations and Pitfalls Enrichment broths should not be used as the sole isolation medium. They are to be used in conjunction with selective and nonselective plating media to increase the probability of isolating pathogens, especially when they may be present in small numbers. Consult references for detailed information and recommended procedures.

6.11.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.12 Preparation of Haemophilus Test Medium (HTM) Agar 6.12.1 Purpose To ensure that the correct validated procedure is followed for the preparation of HTM Agar to provide accurate, reliable and reproducible results having clinical utility.

6.12.2 Introduction Haemophilus Test Medium Agar (HTM Agar) is intended for use in the antimicrobial disc diffusion susceptibility procedure for Haemophilus spp. As described in the Approved Standard M2, published by the National Committee for Clinical Laboratory Standards (NCCLS). In 1966, Bauer, Kirby and others developed a standardized procedure for the antimicrobial susceptibility testing of common, rapidly growing bacteria in which Mueller Hinton Agar was selected as the test medium.This medium in not satisfactory for fastidious organisms such as some streptococci, gonococci and Haemophilus species. Extensive studies performed by Jorgensen and colleagues led to the development of Haemophilus Test Medium (HTM). This medium is Mueller Hinton agar or broth supplemented with X factor (hemin or hematin), V factor (nicotinamide adenine dinucleotide, NAD) and yeast extract. In the test procedure, a standardized suspension of the organism is swabbed over the entire surface of the medium. Paper discs impregnated with specified amounts of antibiotic or other antimicrobial agents are then placed on the surface of the medium, the plate is incubated and zones of the inhibition around each disc are measured. The determination as to whether the organism is susceptible, resistant or intermediate in its response to the agent is made by comparing zone diameters obtained to those provided with NCCLS document M2.

6.12.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of HTM agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations use an evidence based validation process.

6.12.4 Staff Competence Trained qualified competent staff

6.12.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.12.6 Pre-examination Procedures: 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.12.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT HTM Agar Medium Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile tubes with covers Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.12.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.12.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms Refer to Section 5.

6.12.10 Limitations and Pitfalls N/A

6.12.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.13 Preparation of MacConkey Agar 6.13.1 Purpose To ensure that the correct validated procedure is followed for the preparation of MacConkey agar to provide accurate, reliable and reproducible results having clinical utility.

6.13.2 Introduction MacConkey agars are slightly selective and differential plating media mainly used for the detection and isolation of gram-negative organisms. MacConkey Agar is used for isolating and differentiating lactose-fermenting from non lactose-fermenting gram-negative enteric bacilli. Peptones are sources of nitrogen and other nutrients. Lactose is a fermentable carbohydrate. When lactose is fermented, a local pH drop around the colony causes a colour change in the pH indicator (neutral red) and bile precipitation. Bile salts, bile salts no. 3, oxgall and crystal violet are selective agents that inhibit growth of gram-positive organisms. Agar is the solidifying agent.

6.13.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of MacConkey agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.13.4 Staff Competence Trained qualified competent staff

6.13.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.13.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.13.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT MacConkey Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.13.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.13.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Growth + Salmonella enteritidis 13076 18–24 hours No Lactose fermentation 35–37ºC, aerobic, Growth + E. coli 25922 18–24 hours Lactose Fermentation 35–37ºC, aerobic, Growth + Proteus vulgaris 13313 18–24 hours No lactose fermentation 35–37ºC, aerobic, Staph aureus 25923 Growth inhibited 18–24 hours

6.13.10 Limitations and Pitfalls 1. Although MacConkey media are selective primarily for gram-negative enteric bacilli, biochemical and, if indicated, serological testing using pure cultures are recommended for complete identification. Consult appropriate references for further information.

2. Incubation of MacConkey Agar plates under increased CO2 has been reported to reduce the growth and recovery of a number of strains of gram-negative bacilli.

6.13.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.14 Preparation of MacConkey Agar with Sorbitol 6.14.1 Purpose To ensure that the correct validated procedure is followed for the preparation of MacConkey agar with sorbitol to provide accurate, reliable and reproducible results having clinical utility.

6.14.2 Introduction MacConkey Sorbitol Agar and MacConkey II Agar with Sorbitol are selective and differential media for the detection of sorbitol-nonfermenting Escherichia coli serotype O157:H7 associated with hemorrhagic colitis. These media are also referred to as ‘Sorbitol MacConkey Agar’. Escherichia coli serotype O157:H7 is a human pathogen associated with hemorrhagic colitis that results from the action of a shiga-like toxin (SLT). On standard MacConkey Agar containing lactose, this strain is indistinguishable from other lactose-fermenting E. coli. Unlike most E. coli strains, E. coli O157:H7 ferments sorbitol slowly or not at all. Therefore, the efficacy of MacConkey Agar containing sorbitol instead of lactose as a differential medium for the detection of E. coli O157:H7 in stool cultures was determined. Field trial results showed that the growth of E. coli O157:H7 on MacConkey Agar with Sorbitol was heavy and occurred in almost pure culture as colourless sorbitol-nonfermenting colonies. Most organisms of the faecal flora ferment sorbitol and appear pink on this medium. MacConkey Agar with Sorbitol, therefore, permits ready recognition of E. coli O157:H7 in stool cultures. MacConkey Sorbitol Agar and MacConkey II Agar with Sorbitol, modified MacConkey agars using sorbitol instead of lactose, are only slightly selective, since the concentration of bile salts, which inhibits gram-positive microorganisms, is low in comparison with other enteric plating media. Crystal violet also is included in the medium to inhibit the growth of gram-positive bacteria, especially enterococci and staphylococci. MacConkey II Agar with Sorbitol is also formulated to reduce swarming of Proteus species.

6.14.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of MacConkey agar with sorbitol as provided by regional laboratories. It may be adopted/adapted by any laboratory provided that such adaptations use an evidence base validation process.

6.14.4 Staff Competence Trained qualified competent staff

6.14.5 Safety Instructions Follow established laboratory procedures in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.14.6 Pre-examination Procedures 7.1 Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.14.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT MacConkey Agar with sorbitol N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.14.8 Procedure: 1. Prepare according to manufacturer’s instructions 2. Dispense in sterile glassware 3. Perform QC and sterility check 4. Label and store 2–8ºC

6.14.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + E. coli 25922 35–37ºC, aerobic, 18–24 hours Sorbitol fermenter: pink to red Growth + E. coli serotype 0157 35150 35–37ºC, aerobic, 18–24 hours Non-sorbitol fermenter: colourless Growth + Proteus mirabilis 12453 35–37ºC, aerobic, 18–24 hours Non-sorbitol fermenter: colourless

6.14.10 Limitations and Pitfalls 1. It has been reported that some and Pseudomonas aeruginosa are inhibited on MacConkey

Agar when incubated in a CO2-enriched atmosphere. 2. Prolonged incubation of the culture may result in colonies of E. coli serotype O157:H7 losing their characteristic colourless appearance. There are additional species of facultatively anaerobic gram-negative rods that do not ferment sorbitol. 3. The colour of sorbitol-positive colonies can fade, making them hard to distinguish from sorbitol-negative colonies.

6.14.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.15 Preparation of Mueller Hinton (MH) Agar 6.15.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Mueller Hinton Agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.15.2 Introduction Mueller Hinton Agar is recommended for antimicrobial disc diffusion susceptibility testing of common, rapidly growing bacteria by the Kirby-Bauer method, as standardized by the National Committee for Clinical Laboratory Standards (NCCLS). Mueller Hinton Agar was originally developed for the cultivation of pathogenic Neisseria. Bauer, Kirby and others developed a standardized procedure in which Mueller Hinton Agar was selected as the test medium. Mueller Hinton Agar with 5% Sheep Blood is recommended for antimicrobial disc diffusion susceptibility testing of Streptococcus pneumoniae with selected agents; i.e., chloramphenicol, erythromycin, ofloxacin, tetracycline and vancomycin, in addition to oxacillin screening for susceptibility to penicillin, as standardized by the National Committee for Clinical Laboratory Standards (NCCLS). Mueller Hinton Agar and Mueller Hinton II Agar are manufactured to contain low levels of thymine and thymidine and controlled levels of calcium and magnesium. Acid hydrolysate (digest) of casein and beef extract supply amino acids and other nitrogenous substances, minerals, vitamins, carbon and other nutrients to support the growth of microorganisms. Starch acts as a protective colloid against toxic substances that may be present in the medium. Hydrolysis of the starch during autoclaving provides a small amount of dextrose, which is a source of energy. Agar is the solidifying agent.

6.15.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of MH agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.15.4 Staff Competence Trained qualified competent staff

6.15.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.15.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.15.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Mueller Hinton agar 5% sheep blood (MH with blood only) Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.15.8 Procedure 1. Prepare according to manufacturer’s instructions. 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.15.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

E. coli 25922 35–37ºC, aerobic, 18–24 hours Growth +

Staph aureus 25923 35–37ºC, aerobic, 18–24 hours Growth+

Pseudomonas aeruginosa 27853 35–37ºC, aerobic, 18–24 hours Growth +

6.15.10 Limitations and Pitfalls 1. Mueller Hinton agar deeper than 4 mm may cause false resistant results, and agar less than 4 mm deep may be associated with a false-susceptibility report. 2. A pH outside the range of 7.3 ± 0.1 may adversely affect susceptibility test results. If the pH is too low, aminoglycosides and macrolides will appear to lose potency; others may appear to have excessive activity. 3. The opposite effects are possible if the pH is too high.

6.15.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.16 Preparation of Nutrient Agar 6.16.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Nutrient Agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.16.2 Introduction Nutrient Agar is used for the cultivation of bacteria and for the enumeration of organisms in water, sewage, faeces and other materials. Nutrient Agar consists of peptone, beef extract and agar. This relatively simple formulation provides the nutrients necessary for the replication of a large number of microorganisms that are not excessively fastidious. The beef extract contains watersoluble substances including carbohydrates, vitamins, organic nitrogen compounds and salts. Peptones are the principle sources of organic nitrogen, particularly amino acids and longchained peptides. Agar is the solidifying agent.

6.16.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Nutrient agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.16.4 Staff Competence Trained qualified competent staff

6.16.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.16.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.16.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Nutrient agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.16.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.16.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

E. coli 25922 35–37ºC, aerobic, 18–24 hours Growth +

Staph aureus 25923 35–37ºC, aerobic, 18–24 hours Growth +

Strep pyogenes 19615 35–37ºC, aerobic, 18–24 hours Growth+

Pseudomonas aeruginosa 27853 35–37ºC, aerobic, 18–24 hours Growth +

6.16.10 Limitations and Pitfalls N/A

6.16.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.17 Preparation of New York City (NYC) Medium 6.17.1 Purpose To ensure that the correct validated procedure is followed for the preparation of New York City Medium in order to provide accurate, reliable and reproducible results having clinical utility.

6.17.2 Introduction New York City (NYC) Medium, Modified is a semi-transparent selective medium used in qualitative procedures primarily for the isolation of pathogenic Neisseria. Fauer et al., described a new medium, designated NYC medium, for the isolation of pathogenic Neisseria consisting of a peptone-corn starch-agar-phosphate buffered base supplemented with horse plasma, horse hemoglobin, dextrose, yeast dialysate and antimicrobics. The peptones, horse plasma, hemoglobin and enrichment supplement in NYC Medium, Modified provide nutrients for the growth of N. gonorrhoeae and N. meningitidis. The phosphate salts buffer the medium at a neutral pH. This selective medium contains the antimicrobial agents, vancomycin, colistin, anisomycin and trimethoprim, to suppress the normal flora. Vancomycin is active primarily against gram-positive bacteria. Colistin inhibits gram-negative bacteria, including Pseudomonas species, but is not active against Proteus species. Anisomycin inhibits yeasts. Trimethoprim inhibits Proteus.

6.17.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of NYC medium as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.15.4 Staff Competence Trained qualified competent staff

6.17.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.17.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.17.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT New York City medium N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.17.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.17.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Neisseria gonorrhoea 19424 Growth + 18–24 hours 35–37ºC, aerobic, Pseudomonas aeruginosa 27853 Growth - 18–24 hours 35–37ºC, aerobic, Candida albicans 10231 Partial inhibition 18–24 hours 35–37ºC, aerobic, E. coli 25922 Marked to complete inhibition 18–24 hours 35–37ºC, aerobic, Staph epidermidis 12228 Marked to complete inhibition 18–24 hours

6.17.10 Limitations and Pitfalls 1. Vancomycin-resistant GC may be inhibited.

6.17.11 References 1. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.18 Preparation of Peptone Water 6.18.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Peptone Water in order to provide accurate, reliable and reproducible results having clinical utility.

6.18.2 Introduction Peptone Water is used for cultivating non-fastidious organisms, for studying carbohydrate fermentation patterns and for performing the indole test. This nonselective medium has been used as a basal medium for biochemical tests such as carbohydrate fermentation patterns and production of indole. Peptone Water contains peptone as a source of carbon, nitrogen, vitamins and minerals. Sodium chloride maintains the osmotic balance of the medium.

6.18.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Peptone Water as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.18.4 Staff Competence Trained qualified competent staff

6.18.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.18.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.18.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT

Peptone Water N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.18.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.18.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Staph aureus 25923 35–37ºC, aerobic, 18–24 hours Growth +

E. coli 25922 35–37ºC, aerobic, 18–24 hours Growth +

6.18.10 Limitations and Pitfalls N/A

6.18.11 References 1. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.19 Preparation of Phenylethyl Alcohol (PEA) Agar 6.19.1 Purpose To ensure that the correct validated procedure is followed for the preparation of PEA in order to provide accurate, reliable and reproducible results having clinical utility.

6.19.2 Introduction Phenylethyl Alcohol (PEA) Agar is a selective medium for the isolation of gram-positive organisms, particularly gram-positive cocci, from specimens of mixed gram-positive and gram-negative flora. After noting that phenylethyl alcohol exhibited an inhibitory effect on gram-negative bacteria with only slight effect on grampositive organisms, Lilley and Brewer incorporated the chemical in an infusion agar base as a selective agent for the isolation of gram- positive bacteria. Phenylethyl Alcohol Agar, unsupplemented or supplemented with 5% sheep blood, is used in the microbiology laboratory to inhibit gram-negative bacteria, particularly Proteus, in specimens containing a mixed bacterial flora. Phenylethyl Alcohol Agar and Phenylethyl Alcohol Agar with 5% Sheep Blood support the growth of gram-positive bacterial species, due to the content of peptones, which supply nitrogen, carbon, sulfur and trace nutrients. Sodium chloride maintains osmotic equilibrium. Sheep blood is a source of growth factors. Phenylethyl alcohol is bacteriostatic for gram-negative bacteria since it selectively and reversibly inhibits DNA synthesis.

6.19.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of PEA as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.19.4 Staff Competence Trained qualified competent staff

6.19.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.19.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.19.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Phenylethyl Alcohol Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.19.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.19.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Partial to complete Proteus mirabilis 25922 18–24 hours inhibition 35–37ºC, aerobic, Staph aureus 25923 Growth+ 18–24 hours 35–37ºC, aerobic, Growth + Streptococcus pneumoniae 6305 18–24 hours Alpha haemolysis Growth + Streptococcus pyogenes 19615 37ºC, aerobic, 18–24 hours Beta haemolysis

6.19.10 Limitations and Pitfalls 1. Some gram-positive cocci may be slightly inhibited and may require further incubation (to 48hrs) for sufficient growth to be evident. 2. Subculture gram-positive colonies onto Tryptic Soy Agar (TSA) or other biochemical media for definitive identification. 3. Pseudomonas aeruginosa is not inhibited on this medium

6.19.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.20 Preparation of Phenylalanine Agar 6.20.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Phenylalanine Agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.20.2 Introduction Phenylalanine Agar is used for the differentiation of enteric bacilli on the basis of their ability to produce phenylpyruvic acid by oxidative deamination. Ferric Chloride Reagent is used to visualize the phenylalanine deamination reaction. This medium was designed to differentiate members of the Proteeae from other members of the Enterobacteriaceae by the ability of organisms in the genera within the Proteeae to deaminate phenylalanine to phenylpyruvic acid by enzymatic activity. Proteus, Providencia and Morganella species possess this capability. This formula conforms to the modified formula of Ewing et al. Ferric Chloride Reagent is used to determine if a specific microorganism is capable of producing phenylpyruvic acid from phenylalanine. The phenylalanine serves as the substrate for enzymes which are able to deaminate it to form phenylpyruvic acid. The addition of 3–5 drops of a 10% aqueous ferric chloride solution (or a 12% aqueous ferric chloride solution acidified with 2.5 ml of concentrated HCl per 100 ml of reagent) to the cultures following incubation results in the appearance of a light to deep green colour (positive reaction) or no colour change (negative reaction). In a positive reaction, any phenylpyruvic acid present will react with the ferric salt in the reagent to give a green colour.

6.20.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Phenylalanine Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.20.4 Staff Competence Trained qualified competent staff

6.20.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.20.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.20.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Phenylalanine Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.20.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.20.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms.

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Enterobacter aerogenes 13048 Phenylanine deaminase: - 18–24 hours 35–37ºC, aerobic, E. coli 25922 Phenylanine deaminase: - 18–24 hours 35–37ºC, aerobic, Proteus vulgaris 13313 Phenylanine deaminase: + 18–24 hours

+ = colour change from white to green - = no colour change

6.20.10 Limitations and Pitfalls 1. A positive phenylalanine reaction should be interpreted quickly because the green colour disappears within 10 minutes after addition of ferric chloride solution. Adding additional reagent usually regenerates the colour. 2. Certain species rapidly deaminate phenyalanine allowing for a positive test result within 4 hours of incubation.

6.20.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.21 Preparation of Mannitol Salt Agar (MSA) 6.19.1 Purpose To ensure that the correct validated procedure is followed for the preparation of MSA in order to provide accurate, reliable and reproducible results having clinical utility.

6.21.2 Introduction Mannitol Salt Agar is used for the selective isolation and enumeration of staphylococci from clinical and non-clinical materials. Koch, in 1942, reported that only staphylococci grow on agar media containing 7.5% sodium chloride. Chapman further studied this phenomenon in greater detail and concluded that the addition of 7.5% sodium chloride to phenol red mannitol agar results in an improved medium for the isolation of plasma coagulating staphylococci. Mannitol Salt Agar is a nutritive medium due to its content of peptones and beef extract, which supply essential growth factors, such as nitrogen, carbon, sulfur and trace nutrients. The 7.5% concentration of sodium chloride results in the partial or complete inhibition of bacterial organisms other than staphylococci. Mannitol fermentation, as indicated by a change in the phenol red indicator, aids in the differentiation of staphylococcal species.

6.21.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Mannitol Salt Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.21.4 Staff Competence Trained qualified competent staff

6.21.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.21.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.21.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Mannitol Salt Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper 6.21.8 Procedure: 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.21.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Growth+ Staph aureus 25923 18–24 hours Colony colour: Yellow 35–37ºC, aerobic, Growth+ Staph epidermidis 12228 18–24 hours Colony colour: Red 35–37ºC, aerobic, Marked to complete E. coli 25922 18–24 hours inhibition

6.21.10 Limitations and Pitfalls N/A

6.21.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.22 Preparation of Sabouraud Dextrose Agar 6.22.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Sabouraud’s Dextrose agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.22.2 Introduction Sabouraud Dextrose Agar is used in qualitative procedures for cultivation of pathogenic and nonpathogenic fungi, particularly dermatophytes. The medium is rendered more selective for fungi by the addition of antimicrobics. Sabouraud Dextrose Agar is a general-purpose medium devised by Sabouraud for the cultivation of dermatophytes. The low pH of approximately 5.6 is favorable for the growth of fungi, especially dermatophytes, and slightly inhibitory to contaminating bacteria in clinical specimens. The addition of antimicrobials is a modification designed to increase bacterial inhibition. Sabouraud dextrose media are peptone media supplemented with dextrose to support the growth of fungi. Media are also provided with maltose substituted for the dextrose. Peptones are sources of nitrogenous growth factors. The carbohydrate provides an energy source for the growth of microorganisms. Gentamicin is an aminoglycoside antibiotic that inhibits the growth of gram- negative bacteria. Chloramphenicol is inhibitory to a wide range of gram-negative and gram-positive bacteria, and cycloheximide is an antifungal agent that is primarily active against saprophytic fungi and does not inhibit yeasts or dermatophytes.

6.22.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Sabouraud Dextrose Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.22.4 Staff Competence Trained qualified competent staff

6.22.5 Safety Instructions Wear suitable protective clothing.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.22.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.22.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Sabouraud Dextrose Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.22.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.22.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Candida albicans 10231 25–33ºC, aerobic, up to 7 days Growth +

Trichophyton 9533 25–33ºC, aerobic, up to 7 days Growth + mentagrophytes

6.22.10 Limitations and Pitfalls 1. Some fungi may be inhibited by the acidic pH of the medium and by the antimicrobics in the selective media. 2. Avoid overheating a medium with acidic pH because this often causes a soft medium.

6.22.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.23 Preparation of Thayer Martin (TM) Agar 6.23.1 Purpose To ensure that the correct validated procedure is followed for the preparation of TM agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.23.2 Introduction Thayer-Martin Selective Agar and Modified Thayer-Martin (MTM II) Agar are used for the isolation of pathogenic Neisseria from specimens containing mixed flora of bacteria and fungi. Thayer-Martin Selective Agar was developed for the primary isolation of N. gonorrhoeae and N. meningitidis from specimens containing mixed flora taken from the throat, vagina, rectum and urethra. Consisting of Chocolate II Agar with vancomycin, colistin and nystatin, it is formulated to minimize the overgrowth of gonococci and meningococci by contaminants, to suppress the growth of saprophytic Neisseria species and to enhance the growth of pathogenic Neisseria. Martin et al. modified Thayer-Martin Selective Agar by adding trimethoprim to produce Modified Thayer-Martin Agar. A significantly greater number of positive gonococcal isolates from clinical specimens were reported as compared with Thayer-Martin Selective Agar due to the inhibition of swarming Proteus species. Because of its improved performance, it is recommended over earlier formulations for the isolation of N. gonorrhoeae. Thayer-Martin Selective Agar and Modified Thayer-Martin (MTM II) Agar contains an improved GC Agar base, bovine hemoglobin and an enrichment supplement. The GC base contains nitrogenous nutrients in the form of casein and meat peptones, phosphate buffer to maintain pH and corn starch, which neutralizes toxic fatty acids that may be present in the agar. Hemoglobin provides X factor (hemin) for Haemophilus spp. The enrichment supplement provides V factor (nicotinamide adenine dinucleotide, NAD) for Haemophilus species and vitamins, amino acids, coenzymes, dextrose, ferric ion and other factors which improve the growth of pathogenic Neisseria. These selective media contain the antimicrobial agents, vancomycin, colistin and nystatin (V-C-N inhibitor) to suppress the normal flora. Vancomycin is active primarily against gram-positive bacteria. Colistin inhibits gram negative bacteria, including Pseudomonas spp., but is not active against Proteus spp. Nystatin inhibits fungi. Modified Thayer-Martin (MTM II) Agar also contains trimethoprim for the inhibition of Proteus spp.

6.23.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Thayer Martin Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.23.4 Staff Competence Trained qualified competent staff

6.23.5 Safety Instructions 1. Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container slightly closed. 2. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.23.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.23.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT GC Agar Base Bovine Haemoglobin Distilled Water Enrichment Supplement Autoclave V-C-N/V-C-N-T Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.23.8 Procedure: 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.23.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED

E. coli 25922 same conditions Growth -

35–37ºC, aerobic, Neisseria gonorrhoea 19424 Growth + 18-24 hours 35–37ºC, aerobic, Pseudomonas aeruginosa 27853 Growth - 18-24 hours 25–33ºC, aerobic, Candida albicans 10231 Growth - 18-24 hours 35–37ºC, aerobic, Proteus vulgaris 13313 Growth - 18-24 hours

6.23.10 Limitations and Pitfalls Selective media for pathogenic Neisseria may inhibit other pathogenic bacteria, e.g. Haemophilus. The existence of strains of N. gonorrhoeae inhibited by the components of V-C-N Inhibitor and trimethoprim lactate have been reported. While ‘saprophytic’ Neisseria are generally suppressed by selective media, the occasional recovery of N. lactamica on Thayer-Martin Selective Agar has been reported. Some strains of Capnocytophaga species may grow on these selective media when inoculated with oropharyngeal specimens.

6.23.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003

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6.24 Preparation of Xylose Lysine Deoxycholate (XLD) Agar 6.24.1 Purpose To ensure that the correct validated procedure is followed for the preparation of XLD agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.24.2 Introduction XLD Agar is the complete Xylose Lysine Desoxycholate Agar, a moderately selective medium recommended for isolation and differentiation of enteric pathogens, especially Shigella species. XLD Agar was developed by Taylor in order to increase the efficiency of the isolation and identification of enteric pathogens, particularly Shigella. The pathogens are differentiated not also from many nonpathogens which do not ferment lactose or sucrose. Additionally, the medium was formulated to increase the frequency of growth of the more fastidious pathogens, which in other formulations have often failed to grow due to the inclusion of excessively toxic inhibitors. Xylose is incorporated into the medium since it is fermented by practically all enterics except for the shigellae, and this property enables the differentiation of Shigella species. Lysine is included to enable the Salmonella group to be differentiated from the nonpathogens since, without lysine, salmonellae rapidly would ferment the xylose and be indistinguishable from nonpathogenic species. After the salmonellae exhaust the supply of xylose, the lysine is attacked via the enzyme, lysine decarboxylase, with reversion to an alkaline pH which mimics the Shigella reaction. To prevent similar reversion by lysine positive coliforms, lactose and sucrose (saccharose) were added to produce acid in excess. To add to the differentiating ability of the formulation, an H2S indicator system, consisting of sodium thiosulfate and ferric ammonium citrate, is included for the visualization of the hydrogen sulphide produced, resulting in the formation of colonies with black centers. The nonpathogenic H2S producers do not decarboxylate lysine; therefore, the acid reaction produced by them prevents the blackening of the colonies. XLD Agar is both a selective and differential medium. It utilizes sodium desoxycholate as the selective agent and, therefore, it is inhibitory to gram-positive microorganisms.

6.24.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of XLD Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.24.4 Staff Competence Trained qualified competent staff

6.24.5 Safety Instructions 1. Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container slightly closed. 2. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.24.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.24.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT XLD Agar Base N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.24.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.24.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + Salmonella enteritidis 13076 35–37ºC, aerobic, 18–24 hours Colony colour: red with black centre Growth + Proteus vulgaris 13313 35–37ºC, aerobic, 18–24 hours Colony colour: red with black centre Growth + Shigella sonnei 25931 35–37ºC, aerobic, 18–24 hours Colony colour: red to pink

Staph aureus 25923 35-37ºC, aerobic, 18–24 hours Growth inhibited Growth+ E. coli 25922 35-37ºC, aerobic, 18–24 hours Colony colour: Yellow

6.24.10 Limitations and Pitfalls 1. Red, false-positive colonies may occur with some Proteus and Pseudomonas species. 2. Incubation in excess of 48 hours may lead to false-positive results. 3. S. paratyphi A, S. choleraesuis, S. pullorum and S. gallinarum may form red colonies without black centers, thus resembling Shigella species. 4. Some Proteus strains will give black-centered colonies on XLD Agar.

6.24.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.25 Preparation of Thiosulphate Citrate Bile Salt Sucrose (TCBS) Agar 6.25.1 Purpose To ensure that the correct validated procedure is followed for the preparation of TCBS agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.25.2 Introduction Thiosulfate Citrate Bile Salts Sucrose Agar (TCBS Agar) is used for the selective isolation of V. cholerae and Vibrio parahaemolyticus from a variety of clinical and nonclinical specimens. Vibrio species are most widely recognized for their role in human intestinal infections.. The isolation of Vibrio species has been enhanced by the development of media which are highly selective for vibrios. TCBS is the primary plating medium universally used for the selective isolation of vibrios that cause cholera, diarrhea and food poisoning. It was developed by Kobayashi et al. who modified the selective medium of Nakanishi. The combination of alkaline peptone water and TCBS Agar is used in many procedures for the isolation of V. cholerae and other Vibrio species from faeces. TCBS Agar is highly selective for the isolation of V. cholerae and V. parahaemolyticus as well as other vibrios. Inhibition of gram-positive bacteria is achieved by the incorporation of oxgall, which is a naturally occurring substance containing a mixture of bile salts, and sodium cholate, a pure bile salt. Sodium thiosulfate serves as a sulfur source and, in combination with ferric citrate, detects hydrogen sulphide production. Saccharose (sucrose) is included as a fermentable carbohydrate for the metabolism of vibrios. The alkaline pH of the medium enhances the recovery of V. cholerae. Thymol blue and bromthymol blue are included as indicators of pH changes.

6.25.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of TCBS Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.25.4 Staff Competence Trained qualified competent staff

6.25.5 Safety Instructions 1. Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container slightly closed. 2. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.25.6 Pre-examination Procedures: 1. Storage: Store the dehydrated medium below 30OC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.25.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT TCBS Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.25.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.25.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Proteus vulgaris 13313 35ºC, aerobic, 18–24 hours Growth Inhibited

E. coli 25922 35ºC, aerobic, 18–24 hours Growth Inhibited

Streptococcus faecalis 29212 35ºC, aerobic, 18–24 hours Growth Inhibited

Growth + Vibrio parahaemolyticus 17802 35ºC, aerobic, 18–24 hours Colony colour: blue

6.25.10 Limitations and Pitfalls 1. On initial isolation, V. parahaemolyticus may be confused with Aeromonas hydrophila, Plesiomonas shigelloides and Pseudomonas species. 2. Sucrose-fermenting Proteus species produce yellow colonies which may resemble those of Vibrio. 3. TCBS is an unsatisfactory medium for oxidase testing of Vibrio spp. 4. A few strains of V. cholerae may appear green or colourless on TCBS due to delayed sucrose fermentation.

6.25.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.26 Preparation of Trichosel Broth 6.26.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Trichosel Broth in order to provide accurate, reliable and reproducible results having clinical utility.

6.26.2 Introduction Trichosel Broth, Modified is used for the isolation and cultivation of Trichomonas species. Trichosel Broth, Modified is a modification of the Simplified Trypticase™ Serum (STS) Medium of Kupferberg et al. for the cultivation of Trichomonas spp. The classical formula has been modified by the addition of beef extract and horse serum and an increased amount of yeast extract to improve performance. Chloramphenicol, a relatively stable antibiotic, replaces the penicillin and streptomycin recommended for addition to the STS base. Trichosel Broth, Modified contains casein peptone, cysteine, beef extract and yeast extract as sources of amino acids, nitrogen, sulphur, carbon, vitamins and trace ingredients. Maltose is an energy source for the metabolism of microorganisms including Trichomonas spp. Chloramphenicol is a broad spectrum antibiotic which inhibits a wide range of gram-positive and gram-negative bacteria. Horse serum is added to provide growth factors required by Trichomonas.

6.26.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Trichosel Broth as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.26.4 Staff Competence Trained qualified competent staff

6.26.5 Safety Instructions Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.26.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.26.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Trichosel Broth 5% Horse Serum Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.26.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.26.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

C. albicans 10231 35ºC +/- 2ºC for 5 days Good recovery

S. aureus 25923 35ºC +/- 2ºC for 5 days Partial to complete inhibition

Good recovery: Microscopic Trichomonas vaginalis 30001 35ºC +/- 2ºC for 5 days examination reveals typical morphology and motility

6.26.10 Limitations and Pitfalls N/A

6.26.11 References 1. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.27 Preparation of Tetrathionate Broth 6.27.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Tetrathionate Broth in order to provide accurate, reliable and reproducible results having clinical utility.

6.27.2 Introduction Tetrathionate Broth Base is used as a selective enrichment for the cultivation of Salmonella species that may be present in small numbers and compete with intestinal flora. Tetrathionate Broth was originally described by Mueller who found that the medium selectively inhibited coliforms, thereby permitting enteric pathogens to grow virtually without restriction. Kaufmann modified Mueller’s medium and achieved a higher percentage of isolates. The medium is specified in standard methods. Peptones provide nitrogen, vitamins, amino acides and carbon. Oxgall inhibits gram-positive microorganisms. Tetrathionate, which is formed in the medium by the addition of the iodineiodide solution, inhibits the normal intestinal flora of fecal specimens. Calcium carbonate neutralizes and absorbs toxic metabolites.

6.27.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Tetrathionate Broth as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.27.4 Staff Competence Trained qualified competent staff

6.27.5 Safety Instructions 1. Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container slightly closed. 2. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.27.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.27.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Tetrathionate Broth Iodine Solution Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.27.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.27.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Little or no increase in E. coli 25922 35–37ºC, aerobic, 18–24 hours number of colonies

Salmonella typhimurium 14028 35–37ºC, aerobic, 18–24 hours Growth +

6.27.10 Limitations and Pitfalls Enrichment broths should not be used as the sole isolation medium. They are to be used in conjunction with selective and nonselective plating media to increase the probability of isolating pathogens, especially when they may be present in small numbers.

6.27.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.28 Preparation of Candida Bromcresol Green (BCG) Agar 6.28.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Candida Bromcresol Green Agar order to provide accurate, reliable and reproducible results having clinical utility.

6.28.2 Introduction Candida Bromcresol Green (BCG) Agar is a differential and selective medium used for primary isolation and detection of Candida species from clinical specimens. Harold and Snyder demonstrated that the triphenyltetrazolium chloride (TTC) being used as an indicator in Pagano Levin medium retarded the growth of some species of Candida and completely inhibited the growth of others. To overcome this, they replaced TTC with bromcresol green, a non-toxic indicator, to develop Candida BCG Agar. Neomycin is incorporated to inhibit gram- negative and some gram-positive bacteria. This medium consists of peptone agar base supplemented with yeast extract and dextrose to provide the nutrients necessary to support growth. Neomycin is an aminoglycoside antibiotic that is active against aerobic and facultatively anaerobic gram negative bacteria and certain gram- positive species. Bromcresol green aids in differentiation and identification of Candida species based on dextrose fermentation. A change in the pH causes the medium to become a yellow colour around the colonies of organisms that ferment dextrose.

6.28.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Candida Bromcresol Green Agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.28.4 Staff Competence Trained qualified competent staff

6.28.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.28.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.28.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT BCG Agar Base N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.28.8 Procedure: 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.28.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + C. albicans 10231 30ºC +/– 2ºC for 24–72 hours Yellow coloration Growth + C. tropicalis 9968 30ºC +/– 2ºC for 24–72 hours Yellow coloration Growth Inhibited E. coli 25922 30ºC +/– 2ºC for 24–72 hours Green coloration

6.28.10 Limitations and Pitfalls Since the nutritional requirements of yeast vary, some strains may be encountered that fail to grow or grow poorly on this medium.

6.28.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.29 Preparation of CHROMagar 6.29.1 Purpose To ensure that the correct validated procedure is followed for the preparation of CHROMagar in order to provide accurate, reliable and reproducible results having clinical utility.

6.29.2 Introduction CHROMagar S. aureus medium is designed for the isolation and identification of aureus without the use of confirmatory testing from clinical sources. With the inclusion of chromogenic substrates in the medium, colonies of S. aureus produce a mauve colour, thus allowing identification on the primary isolation plate. Specially selected peptones supply the nutrients. The chromogen mix consists of artificial substrates (chromogens), which release an insoluble coloured compound when hydrolyzed by specific enzymes. Selective agents have also been added for the suppression of gram-negative organisms and partial suppression of yeast.

6.29.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of CHROMagar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.29.4 Staff Competence Trained qualified competent staff

6.29.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.29.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.29.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT CHROMagar Base N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Petri Dishes Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.29.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.29.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS Growth + C. albicans 10231 30ºC +/– 2ºC for 24–72 hours Green coloration Growth + C. tropicalis 9968 30ºC +/– 2ºC for 24–72 hours Blue coloration Growth + C. krusei 34135 30ºC +/– 2ºC for 24–72 hours Pink coloration

6.29.10 Limitations and Pitfalls 1. Occasional strains of coagulase negative staphylococci such as S. cohnii, S. intermedius and S. schleiferi, as well as corynebacteria and yeasts, may produce mauve-coloured colonies at 24hours. Differentiation of S. aureus from non-S. aureus can be accomplished by coagulase, other biochemicals or Gram stain. Resistant gram-negative bacilli, which typically appear as small blue colonies, may also breakthrough. 2. Incubation beyond 24 hours is not recommended due to an increase in potential false positives.

6.29.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.30 Preparation of Lim Broth 6.30.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Lim Broth in order to provide accurate, reliable and reproducible results having clinical utility.

6.30.2 Introduction Lim Broth us used for the selective enrichment of group B streptococci (Streptococcus agalactiae), especially from genital specimens. Since its emergence in the 1970s, neonatal group B streptococcal disease has become the major infectious cause of illness and death among newborns. The use of Todd Hewitt Broth with colistin and nalidixic acid is a medium recommended to maximize the likelihood of recovering group B streptococci upon plating on sheep blood agar. Lim Broth is prepared from Todd Hewitt Broth by the addition of colistin and nalidixic acid, at the recommended concentrations, plus yeast extract for enhanced growth of group B streptococci. Todd Hewitt Broth base is a general-purpose medium primarily used for the cultivation of ß-hemolytic streptococci, especially for serologic studies. The peptones, dextrose and salts provide an excellent nutritional base for the growth of streptococci. The added yeast extract is a rich source of B-complex vitamins. Dextrose stimulates hemolysin production. Disodium phosphate and sodium carbonate provide buffering action to counteract the acidity produced during the fermentation of the carbohydrate, thereby protecting the haemolysin from inactivation by the acid. Nalidixic acid and colistin suppress growth of gram-negative bacteria.

6.30.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Lim Broth as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.30.4 Staff Competence Trained qualified competent staff

6.30.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.30.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ªC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.30.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Lim Broth N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper 6.30.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.30.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

S. agalactiae 13813 35–37ºC, aerobic, 18–24 hours Turbidity

Uninoculated Broth N/A 35–37ºC, aerobic, 18–24 hours No turbidity

6.30.10 Limitations and Pitfalls N/A

6.28.11 References 1. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.31 Preparation of Kligler Iron Agar (KIA) 6.31.1 Purpose To ensure that the correct validated procedure is followed for the preparation of KIA in order to provide accurate, reliable and reproducible results having clinical utility.

6.31.2 Introduction Kligler Iron Agar is used for the differentiation of members of the Enterobacteriaceae on the basis of their ability to ferment dextrose and lactose and to liberate sulphides. Kligler developed a simple lead acetate medium for the differentiation of the typhoid-paratyphoid group. Kligler Iron Agar, in addition to casein and meat peptones, contains lactose and dextrose which enable the differentiation of species of enteric bacilli due to colour changes of the phenol red pH indicator in response to the acid produced during the fermentation of these sugars. The dextrose concentration is only 10% of the lactose concentration. The combination of ferric ammonium citrate and sodium thiosulfate enables the detection of hydrogen sulfide production. Lactose nonfermenters (e.g., Salmonella and Shigella) initially produce a yellow slant due to acid produced by the fermentation of the small amount of dextrose. When the dextrose supply is exhausted in the aerobic environment of the slant, the reaction reverts to alkaline (red slant) due to oxidation of the acids. The reversion does not occur in the anaerobic environment in the butt, which remains acid (yellow butt). Lactose fermenters produce yellow slants and butts because enough acid is produced in the slant to maintain an acid pH under aerobic conditions. Organisms incapable of fermenting either carbohydrate produce red slants and butts. Hydrogen sulfide production is evidenced by a black colour either throughout the butt, or in a ring formation near the top of the butt. Gas production (aerogenic reaction) is detected as individual bubbles or by splitting or displacement of the agar.

6.31.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of KIA as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.31.4 Staff Competence Trained qualified competent staff

6.31.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.31.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.31.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT KIA N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.31.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.31.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Slant A, Butt A E. coli 25922 18–24 hours Gas +, H2S - 35–37ºC, aerobic, Slant K, Butt A Proteus vulgaris 6380 18–24 hours Gas -, H2S + 35–37ºC, aerobic, Slant K, Butt A Salmonella enteritidis 13076 18–24 hours Gas +, H2S + 35–37ºC, aerobic, Slant A, Butt A Shigella flexneri 12022 18–24 hours Gas +, H2S + 35–37ºC, aerobic, Slant A, Butt A Citrobacter freundii 8090 18–24 hours Gas +, H2S +

A = Acid = Yellow K = Alkaline = No colour change

6.31.10 Limitations and Pitfalls

1. H2S producing organisms may produce a black precipitate to such a degree that the reaction in the butt is

completely masked. If H2S is produced dextrose is fermented even if it is not observed. 2. Further biochemical test and serological typing must be performed for definite identification and confirmation of organisms. 3. Best reactions are obtained on freshly prepared medium.

6.31.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.32 Preparation of Urea 6.32.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Urea in order to provide accurate, reliable and reproducible results having clinical utility.

6.32.2 Introduction Urea Agar and Urease Test Broth are used for the differentiation of organisms, especially the Enterobacteriaceae, on the basis of urease production. Urea Agar was devised by Christensen for use as a solid medium for the differentiation of enteric bacilli. Urease Test Broth was developed by Rustigian and Stuart. The base is also supplied as a filter-sterilized 10_ concentrated solution in tubes for use in preparing Urea Agar slants and broth in the laboratory. The urea medium of Rustigian and Stuart is particularly suited for the differentiation of Proteus species from other gram negative enteric bacilli capable of utilizing urea; the latter are unable to do so in Urease Test Broth because of limited nutrients and the high buffering capacity of the medium. To provide a medium with greater utility, Urea Agar was devised by Christensen with peptone and dextrose included and reduced buffer content to promote more rapid growth of many of the Enterobacteriaceae and permit a reduction in incubation time. Yeast extract provides vitamins and cofactors required for growth and as an additional source of nitrogen and carbon. Dextrose is included as an energy source. Sodium chloride maintains the osmotic balance of the medium. Potassium phosphate monobasic and dibasic provides buffering capability. When organisms utilize urea, ammonia is formed during incubation which makes the reaction of these media alkaline, producing a red-pink colour. Consequently, urease production may be detected by the change in the phenol red indicator.

6.32.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Urea as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.32.4 Staff Competence Trained qualified competent staff

6.32.5 Safety Instructions 1. Irritating to eyes, respiratory system and skin. Avoid contact with skin and eyes. Do not breathe dust. Wear suitable protective clothing. Keep container slightly closed. 2. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.32.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.32.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Urea (Broth/Agar) Base N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.32.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.32.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Growth + Enterobacter aerogenes 13048 35–37ºC, aerobic, 18–24 hours Urease - Growth + E. coli 25922 35–37ºC, aerobic, 18–24 hours Urease - Growth + Proteus vulgaris 13315 35–37ºC, aerobic, 18–24 hours Urease + Growth+ Salmonella typhimurium 14028 35–37ºC, aerobic, 18–24 hours Urease -

Urease + = pink coloration of media Urease – = No colour change

6.32.10 Limitations and Pitfalls Urea agar 1. The alkaline reaction produced in this medium after prolonged incubation may not be caused by urease activity. False positive reactions may occur due to the utilization of peptones (especially in slant agar by Pseudomonas aeruginosa, for example) or other proteins which raise the pH due to protein hydrolysis and the release of excessive amino acid residues. To eliminate possible protein hydrolysis, perform a control test with the same test medium without urea. 2. Do not heat or reheat the medium because urea decomposes very easily. 3. Urea Agar detects rapid urease activity of only the urease positive Proteus species. For results to be valid for the detection of Proteus, the results must be read within the first 2-6 hours after incubation. Urease-positive Enterobacter, Citrobacter or Klebsiella, in contrast, hydrolyze urea much more slowly, showing only slight penetration of the alkaline reaction into the butt of the medium in 6 hours and requiring 3-5 days to change the reaction of the entire butt.

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6.33 Preparation of Simmons Citrate 6.33.1 Purpose To ensure that the correct validated procedure is followed for the preparation of Simmons Citrate in order to provide accurate, reliable and reproducible results having clinical utility.

6.33.2 Introduction Simmons Citrate Agar is used for the differentiation of gram negative bacteria on the basis of citrate utilization. Koser, in 1923, developed a liquid medium consisting of inorganic salts in which an ammonium salt was the only source of nitrogen and citrate was the sole carbon source in order to differentiate between what are now known as coli and Enterobacter aerogenes as part of the IMViC (Indole- Methyl Red-Voges Proskauer-Citrate) reactions. Simmons, in 1926, modified Koser’s formulation with the addition of 1.5% agar and bromthymol blue. Organisms capable of metabolizing citrate grow well on this medium. Organisms able to utilize ammonium dihydrogen phosphate and sodium citrate as the sole sources of nitrogen and carbon respectively, will grow on this medium and produce an alkaline reaction as evidenced by a change in the colour of the bromthymol blue indicator from green (neutral) to blue (alkaline).

6.33.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of Simmons Citrate as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.33.4 Staff Competence Trained qualified competent staff

6.33.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.33.6 Pre-examination Procedures 7.1 Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.33.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT Simmons Citrate Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.33.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.33.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

35–37ºC, aerobic, Citrate utilization: + Enterobacter aerogenes 13048 18–24 hours Colour of medium: blue 35–37ºC, aerobic, Citrate utilization: - E. coli 25922 18–24 hours Colour of medium: green 35–37ºC, aerobic, Citrate utilization: + Salmonella enteritidis 13076 18–24 hours Colour of medium: blue 35–37ºC, aerobic, Shigella flexneri 9199 Complete inhibition 18–24 hours

6.33.10 Limitations and Pitfalls 1. When inoculating a variety of biochemicals, flame the inoculating loop or needle before streaking Simmons Citrate Agar or inoculate the agar first to avoid a false positive result. 2. Some citrate positive organisms require 48 hours or longer incubation for a pH change to occur.

6.33.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.34 Preparation of MILS Medium 6.34.1 Purpose To ensure that the correct validated procedure is followed for the preparation of MILS Medium in order to provide accurate, reliable and reproducible results having clinical utility.

6.34.2 Introduction MILS (Motility Indole Lysine Sulphide) Medium is used for differentiating Enterobacteriaceae based on motility, lysine decarboxylation and deamination, indole and sulphide production. Enzymatic digests of gelatin, casein, animal tissue and heart infusion supply amino acids and other complex nitrogenous substances. Yeast extract is added to MILS Medium primarily to supply the B-complex vitamins. Dextrose is a source of energy. A small amount of agar is added for demonstration of motility along a stab line of inoculation. Growth of motile organisms extends out from the line of inoculation, while nonmotile organisms grow only along the stab line. The pH indicator bromcresol purple is used in MILS Medium to facilitate detection of decarboxylase activity. When inoculated with an organism that ferments dextrose, acids are produced that lower the pH, causing the indicator in the medium to change from purple to yellow. The acidic pH also stimulates enzyme activity. Organisms that possess a specific decarboxylase degrade the amino acid provided in the medium, yielding a corresponding amine. Lysine decarboxylation yields cadaverine. The production of these amines elevates the pH and causes the medium in the bottom portion of the tube to return to a purple colour. The medium in the upper portion of the tube remains acidic because of the higher oxygen tension. If the organism being tested does not produce the required decarboxylase, the medium remains yellow (acidic) throughout or yellow with a purple or red reaction near the top. Lysine deamination produces a colour change in the upper portion of MILS Medium. Oxidative deamination of lysine yields a compound that reacts with ferric ammonium citrate, producing a burgundy red colour in the top centrimeter of the medium.3 (The bottom portion of the medium remains acidic.) This reaction can only be detected if lysine decaraboxylase is not produced, which is the case with Proteus, Morganella and Providencia species. Indole is produced in MILS Medium by organisms that possess the enzyme tryptophanase. Tryptophanase degrades the typtophan present in the casein peptone, yielding indole. Indole can be detected in the medium by adding Kovacs’ reagent to the agar surface. The indole combines with the p-dimethylaminobenzaldehyde of Kovacs’ reagent and produces a red complex. MILS Medium is also used in the demonstration of hydrogen sulfide production. Hydrogen sulfide, which is produced by some enteric organisms from sulfur compounds contained in the medium, reacts with ferric ion, producing a characteristic black precipitate.

6.34.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of MILS Medium as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.34.4 Staff Competence Trained qualified competent staff

6.34.5 Safety Instructions: 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

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6.34.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

6.34.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT MILS Medium N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.34.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.34.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Motility +Indole + 35–37ºC, aerobic, E. coli 25922 Lysine deaminase + 18–24 hours Lysine decarboxylase - Motility + 35–37ºC, aerobic, Indole - Proteus mirabilis 25933 18–24 hours Lysine deaminase + Lysine decarboxylase - Motility + 35–37ºC, aerobic, Indole - Salmonella enteritidis 13076 18–24 hours Lysine deaminase - Lysine decarboxylase + Motility - 35–37ºC, aerobic, Indole – (+) Shigella flexneri 12022 18–24 hours Lysine deaminase - Lysine decarboxylase -

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6.34.10 Limitations and Pitfalls 1. Do not add reagent Kovacs until the final lysine deaminase, lysine decarboxylase and motility results have been interpreted. 2. Occasionally, the indole test produces false negative or falsely weak reactions.

6.34.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003.

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6.35 Preparation of Triple Sugar Iron (TSI) Agar 6.35.1 Purpose To ensure that the correct validated procedure is followed for the preparation of TSI agar in order to provide accurate, reliable and reproducible results having clinical utility.

6.35.2 Introduction Triple Sugar Iron Agar (TSI Agar) is used for the differentiation of gram-negative enteric bacilli based on carbohydrate fermentation and the production of hydrogen sulfide. Hajna developed the formulation for TSI Agar by adding sucrose to the double sugar (dextrose and lactose) formulation of Kligler Iron Agar. The addition of sucrose increased the sensitivity of the medium by facilitating the detection of sucrose-fermenting bacilli, as well as lactose and/or dextrose fermenters. TSI Agar contains three sugars (dextrose, lactose and sucrose), phenol red for detecting carbohydrate fermentation and ferrous ammonium sulfate for detection of hydrogen sulfide production (indicated by blackening in the butt of the tube). Carbohydrate fermentation is indicated by the production of gas and a change in the colour of the pH indicator from red to yellow. To facilitate the detection of organisms that only ferment dextrose, the dextrose concentration is one-tenth the concentration of lactose or sucrose. The small amount of acid produced in the slant of the tube during dextrose fermentation oxidizes rapidly, causing the medium to remain red or revert to an alkaline pH. In contrast, the acid reaction (yellow) is maintained in the butt of the tube because it is under lower oxygen tension. After depletion of the limited dextrose, organisms able to do so will begin to utilize the lactose or sucrose. To enhance the alkaline condition of the slant, free exchange of air must be permitted by closing the tube cap loosely. If the tube is tightly closed, an acid reaction (caused solely by dextrose fermentation) will also involve the slant.

6.35.3 Scope The procedure provides detailed instructions for microbiology services for the preparation of TSI agar as offered by regional laboratories. It may be adapted/adopted by any laboratory as needed, provided that such adaptations used an evidence based validation process.

6.35.4 Staff Competence Trained qualified competent staff

6.35.5 Safety Instructions 1. Follow established laboratory procedure in handling and disposing of infectious materials.

Follow established laboratory procedures in handling and disposing of infectious materials.

Please refer to CRM-SOP 20: Safety Guidelines for the Microbiology Laboratory.

6.35.6 Pre-examination Procedures 1. Storage: Store the dehydrated medium below 30ºC. The dehydrated medium is very hydroscopic. Keep container closed.

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6.35.7 Table of media, reagents, materials and equipment

MEDIA REAGENTS MATERIALS/EQUIPMENT

TSI Agar N/A Distilled Water Autoclave Balance Hotplate/Stove Conical Flask Measuring Cylinder Weighing boats/paper Sterile glassware Incubator Spirit Lamp/Bunsen Burner pH meter/paper

6.35.8 Procedure 1. Prepare according to manufacturer’s instructions 2. Dispense into appropriate glassware 3. Perform QC and sterility check 4. Store at 2–8ºC

6.35.9 Quality Control Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organisms

ORGANISM ATCC NUMBER INCUBATION CONDITIONS EXPECTED RESULTS

Slant A, Butt A E. coli 25922 35–37ºC, aerobic, 18–24 hours Gas +, H2S - Slant K, Butt A Proteus vulgaris 6380 35–37ºC, aerobic, 18–24 hours Gas -, H2S + Slant A, Butt A Shigella flexneri 12022 35–37ºC, aerobic, 18–24 hours Gas -, H2S - Slant A, Butt A Citrobacter freundii 8090 35–37ºC, aerobic, 18–24 hours Gas +, H2S + Slant K, Butt A Salmonella typhimurium 14028 35–37ºC, aerobic, 18–24 hours Gas +, H2S +

A = Acid = Yellow K = Alkaline = No colour change

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6.35.10 Limitations and Pitfalls 1. Hydrogen sulfide production may be evident on Kligler Iron Agar but negative on Triple Sugar Iron Agar. Studies by Bulmash and Fulton showed that the utilization of sucrose could suppress the enzymatic mechanisms

responsible for H2S production. Padron and Dockstader found that not all H2S-positive Salmonella are positive on TSI. 2. Sucrose is added to TSI to eliminate some sucrose-fermenting lactose-nonfermenters such as Proteus and Citrobacter spp. 3. Further biochemical tests and serological typing must be performed for definite identification and confirmation of organisms. 4. Do not use an inoculating loop to inoculate a tube of Triple Sugar Iron Agar. While stabbing the butt, mechanical splitting of the medium occurs, causing a false positive result for gas production.

6.35.11 References 1. Difco Manual 11th edition. 1998. DifcoLaboratories. Division of Becton Dickinson and Co. 2. Zimbro, M; Power, D ed. Difco and BBL Manual. 2003

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7.0 References Manual of Clinical Microbiology American Society for Microbiology Press 7th Edition

Difco Manual 11th Edition Bicton Dickinson

NCCLS Volume 23 (M2-A8) January 2003

Quality Assurance Principles and Practice I the Microbiology Laboratory Edited by JJS Snell, DFJ Brown and C Roberts Public Health Laboratory 1999

Clinical and Pathogenic Microbiology 2nd Edition Barbara J Howard, John F Keiser, Thomas F Smith, Alice S Weissfeld and Richard C Tilton 1994

BBL Quality Control and Product Information Manual for Plated Media July 1994

The Hi Media Manual for Microbiology Practice 1998

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Appendix 1: Preparation of Media (Flowchart)

Dehydrated media Distilled/Deionized Water

Record Lot# Dissolve media as per Expiration date manufacturer’s instructions

Sterilize appropriately

Cool to 50–56ºC

Aseptically add any supplement or additives if necessary

Aseptically dispense in Petri-dishes, or tubes

Label

Do sterility check and Quality Control

Store Discard appropriately Contamination: Sterile: Does not Support Corrective support growth growth of action of organism organism

Record Results Distribute to laboratory for use

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Appendix 2: Preparation of Suspensions for Quality Control of Nonexempt Media

working control (wc)

prepare suspension of wc to match a 0.5 McFarland standard

nonselective media selective media

dilute suspension 1:100 dilute suspension 1:10 inocultae medium inocultae medium use a 10μl calibrated loop use a 10μl calibrated loop streak for isolation streak for isolation

after appropriate incubation, examine for:

little or no growth of organisms adequate growth and susceptible to inhibitory agents; typical morphology growth of expected organisms

(use a 1:1000 dilution if isolated (use a 1:1000 dilution if isolated colonies are not produced) colonies are not produced)

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Appendix 3: Media Quality Control Record Sheet

QUALITY CONTROL OF MEDIA PERFORMANCE TEST OF CONTROL ORGANISMS

DATE: ......

MEDIA ...... BATCH No: ...... EXP Date: ......

PERFORMED BY: ......

ORGANISM LOT# EXP DATE EXPECTED RESULTS COMMENTS RESULTS

REVIEWED BY:...... DATE: ......

REVIEWED BY:...... DATE: ......

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Appendix 4: Media Quality Control Sheet Approved by Comments action corrective Sterility Check Expiry date Lot # Additive Name pH Media Batch # Volume prepared Expiry Date Lot # Man. Prepared Prepared by Date Media Type Along with the above, Mueller Hinton Agar has additional QC checks

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Appendix 5: Exempt and Non-exempt Categories for Media

Exempt and Nonexempt Categories for Media included in CAP Surveys (1984, 1988, 2001)a (From Jones RN, Krisher K, Bird DS, and the College of American Pathologists Microbiology Resource Committee. Results of the survey of the quality assurance for commercially prepared microbiology media. Arch Path Lab Med. 203;127(6):661-665. Reprinted with permission from the College of American Pathologists.)

CATEGORY EXEMPT c NONEXEMPT d,e

Blood agar General Chocolate agar Nutrient broth bacteriologic media Thioglycolate broth Urease agar

Brain heart infusion (BHI) blood culture broth Biphasic blood culture bottle medium Blood culture media Centrifugation/isolation tubes (adult) Peptone broth Thiol blood culture broth Trypticase soy blood broth

Columbia (CAN) agar Selective media for enterococci with or without azide LIM broth Todd-Hewitt broth Media for Mannitol salt agar Desoxycholate brothe gram-positive Phenylethyl alcohol (PEA) agar Trans-vaginal brothe bacteria Selective agar for Group A Streptococcus Chocolate agar with pyridoxale Sheep blood agar with sulfamethoxaole/trimethoprim (SXT) Enterococcus (Streptococcus) faecalis broth

Cefsulodin irgasan novobiocin (CIN) agar Citrate agar Cystine lactose electrolyte deficient (CLED) agar (EMB) agar Gram-negative (GN) broth Hektoen (HEK) agar Media for MacConkey agar MacConkey sorbitol agar gram-negative Salmonella-Shigella (SS) agar Chocolate agar with bacitracine bacteria Selenite broth Thiosulfate citrate bile salts sucrose (TCBS) agar Triple sugar iron (TSI) agar with sheep blood with ampicillin Xylose lycine desoxycholate (XLD) agar

Martin-Lewis agar Neisseria gonorrhoeae Thayer-Martin agar (modified)c Chocolate agar with IsoVitaleX® (GC) media GC-Lectc,d TM New York City agarc

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CATEGORY EXEMPT c NONEXEMPT d,e

Reagan-Lowe agar Bordetella pertussis media Bordet Gengou agare

Legionella media Legionella selective (CYE/BCYE) agarc,d Selective Legionella agar with DGVPd,e

Burkholderia cepacia (PC) media Pseudomonas cepacia (PC) agarc OFPBL agard,e

Campylobacter blood agar (Blaser) Campylobacter media Charcoal selective agar with CVCd Campylobacter agar with CVAd

Anaerobic blood agar Anaerobic phenylthyl alcohol (PEA) agar Bacteriodes bile esculin (BBE) agar CDC anaerobe 5% sheep blood agar Anaerobic media Brucella agar w/hemin/Vitamin K with PEA Brucella laked blood agar with KVd CDC anaerobe laked blood agar with KVd Egg yolk (modified) agar Kanamycin laked blood agar

AFB biphasic bottle mediumd Mycobacteria (AFB) mediad Lowenstein-Jensen media American Trudeau Society (ATS) agare Middlebrook agar Mitchison’s agare Automated AFB bottle brothsb,d Petragnani mediume

Cornmeal agar Cornmeal agar with Tween Inhibitory mould agar Brain heart infusion with 5% sheep Inhibitory mould agar with gentamicin blood/CCd Soy peptone agar with CC without pH BIGGY agard,e indicatorsd Birdseed agare Fungal media Brain heart infusion agar with 5% Brain heart infusion agar with 5% sheep sheep blood/PSd,e blood/CGd Dermatophyte test mediume Sabourand’s dextrose agar Potato flakes agar with or without Sabourand’s dextrose agar with CGd CCe,e a Exempt: Extrapolated Failure Rate of ≤ 0.5%; Nonexempt: Extrapolated Failure Rate of >0.5%; media with insufficient data for categorization is considered nonexempt and QC is required. b Represents formulations from BD Diagnostic Systems (Sparks, MD) or BioMerieux (Raleigh/Durham, NC). Refer to manufacturer’s package insert for specific QC information. c Quality control of exempt media used for fastidious organisms (in particular exempt media for recovery of N. gonorrhoeae, H. influenzae, Campylobacter sp., Legionella sp., and B. cepacia among others) strongly recommended to ensure optimum recovery of organisms. Refer to Table 3 d Abbreviations: AFB (Acid Fast Bacilli); BIGGY (Bismuth sulfite Glucose Glycien Yeast); CC(Cyclohexmide/Chloramphenicol); CG (Chloramphenicol/Gentamicin); CVA (Cefoperazone/Vancomycin/Amphptericin B);CVC (Cefoperazone/Vancomycin/Cycloheximide); CYE/BCYE (Buffered Charcoal Yeast Extract); DVGP (Dye, Vancomycin, Glycine, Polymyxin B); GC (Gonococcal); KV (Kanamycin/Vancomycin); OFPBL (Oxidative Fermatative Polymyxin B, Bacitracin and Lactose); PS (Penicillin/Streptpmycin) e Media deemed nonexempt because of insufficient data for calculation of extrapolated failure rate.

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